Emeritus Faculty, Acad Council, Chemical and Systems Biology
PhD, Brown University
Insulin is one of the primary regulators of rapid anabolic responses in the body. Defects in the synthesis and/or ability of cells to respond to insulin results in the condition known as diabetes mellitus. To better design methods of treatment for this disorder, we have been focusing our research on how insulin elicits its various biological responses. We are utilizing the techniques of immunology, molecular biology, and biochemistry to study:
(i) How does the insulin receptor initiate the response to insulin? Like various oncogenes, the insulin receptor has an intrinsic enzymatic activity; it phosphorylates various proteins on tyrosine residues. This enzymatic activity has been found to be critical for insulin to elicit its various responses. The receptor kinase tyrosine phosphorylates various endogenous proteins. These proteins bind and activate a lipid kinase called a phosphatidylinositol 3-kinase. This kinase activates a serine/threonine kinase called Akt or PKB. A major focus is to understand the role of this serine kinase in eliciting various biological responses. Novel substrates for this kinase are being isolated and genes regulated by this kinase are being identified.
(ii) How is the response to insulin modulated? Cells from non-insulin dependent diabetics (the most common form of diabetes, ~5 million in the US) exhibit a profound resistance to insulin. This resistance can be mimicked in cell cultures by stimulating the serine phosphorylation of the insulin receptor and/or various substrates of the insulin recpetor tyrosine kinase. We are therefore exploring the hypothesis that excessive serine phosphorylation of the insulin receptor and/or insulin receptor substrates in these individuals causes this insulin resistance. We are determining the serine residues phosphorylated in the receptor and insulin receptor substrates, the enzymes response for this phosphorylation, and the consequences of these phosphorylations;
(iii) How is the response to insulin terminated? We have purified to homogeneity a protease with a high specificity for insulin and capable of cleaving insulin at the same sites as those identified in insulin cleaved in intact cells. We have also isolated the cDNA which encodes for this protease and are overexpressing this protease in mammalian cells to determine whether it will affect the termination of the insulin response; and
(iv) What is the relationship of the insulin receptor to the receptor for other insulin-like growth factors? We are comparing the abilities of these different receptors to stimulate various biological responses.
Muscle wasting in chronic renal failure is associated with increased morbidity and mortality; however, resistance exercise is effective at increasing muscle mass while improving muscle strength and function. To study the mechanism by which this occurs, we compared uremic and control rats where work overload was surgically induced unilaterally in the plantaris muscle. We found that work overload increases muscle insulin-like growth factor-1 and mechano-growth factor expression. This in addition to direct mechanical activation of signaling was likely the cause of the observed increased in the protein levels and phosphorylation of the mediators of these growth factors, the insulin receptor substrate-1/phosphoinositide 3-kinase/Akt pathway. The mechanical enhancement of signal transduction appeared to be mediated in part by increased signal protein levels and decreased SOCS2 mRNA expression (suppressor of cytokine signaling-2). Despite impaired basal signaling, the work-induced signaling response was similar to that observed in nonuremic rats and produced changes consistent with decreased muscle protein degradation, increased protein synthetic capacity, and an increased number of multinucleated muscle cells. Our studies suggest that these work-induced changes account for the improved uremic muscle mass reaching levels comparable to those seen in normal rats.
View details for DOI 10.1038/sj.ki.5002801
View details for Web of Science ID 000254085100011
View details for PubMedID 18216779
cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.
View details for DOI 10.1038/sj.emboj.7601431
View details for Web of Science ID 000242891100008
View details for PubMedID 17124499
Although a number of studies and approaches have indicated that activation of the Ser/Thr kinase called Akt/protein kinase B is critical for the insulin-stimulated increase of glucose uptake in adipocytes, other studies have indicated that this enzyme may play an ancillary role. For example, a recent study indicated that neomycin would allow insulin-stimulated Glut4 translocation and glucose transport in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, a known inhibitor of Akt activation (James, D. J., Salaun, C., Brandie, F. M., Connell, J. M. C., and Chamberlain, L. H. (2004) J. Biol. Chem. 279, 20567-20570). To better understand this observation, we examined a number of downstream targets of Akt. As previously reported, treatment of 3T3-L1 adipocytes with neomycin prevented the wortmannin inhibition of insulin-stimulated glucose transport. However, in the presence of neomycin, wortmannin did not inhibit the insulin-stimulated phosphorylation of several downstream targets of Akt including a proline-rich Akt substrate of 40 kDa, ribosomal protein S6, and glycogen synthase kinase-3. In addition, neomycin did not prevent the ability of a structurally unrelated PI 3-kinase inhibitor, LY294002, to inhibit the insulin-stimulated activation of glucose uptake. Moreover, neomycin reversed the inhibitory effect of wortmannin but not LY294002 on insulin stimulation of Akt kinase activity. Finally, neomycin was found to inactivate in vitro the PI 3-kinase inhibitory actions of wortmannin but not LY294002. These results indicate that the effects of neomycin in adipocytes are not mediated via its ability to sequester phosphatidylinositol 4,5-bisphosphate but are instead caused by the ability of neomycin to inactivate wortmannin.
View details for DOI 10.1074/jbc.M411540200
View details for Web of Science ID 000225960800038
View details for PubMedID 15504741
Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCdelta and PKCtheta correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCdelta, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCdelta or wild-type PKCdelta followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCdelta was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1-glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCdelta acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCdelta on at least 18 sites. The importance of three of the PKCdelta phosphorylation sites in IRS-1 was shown in vitro by a 75-80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCdelta modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
View details for DOI 10.1042/BJ20031493
View details for Web of Science ID 000189290600012
View details for PubMedID 14583092
Expression of constitutively active Akt3 was found to increase the size of MCF-7 cells approximately twofold both in vitro and in vivo. A regulatable version of Akt1 (MER-Akt) was also found capable of inducing a twofold increase in the size of H4IIE rat hepatoma cells. Rapamycin, a specific inhibitor of mTOR function, was found to inhibit the Akt-induced increase in cell size by 70%, presumably via inhibition of the Akt-induced increase in protein synthesis. To determine whether Akt could be inhibiting protein degradation, thereby contributing to its ability to induce an increase in cell size, we conducted protein degradation experiments in the H4IIE cell line. Activation of MER-Akt was found to inhibit protein degradation to a degree comparable to insulin treatment. The effects of these two agents on protein degradation were not additive, thereby suggesting that they were acting on a similar pathway. An inhibitor of the phosphatidylinositol 3-kinase pathway, LY-294002, blocked both insulin- and Akt-induced inhibition of protein degradation, again consistent with the hypothesis that both agents were acting on the same pathway. In contrast, rapamycin did not block the ability of either agent to inhibit protein degradation. These results indicate that Akt increases cell size through both mTOR-dependent and -independent pathways and that the latter involves inhibition of protein degradation. These studies are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via Akt.
View details for DOI 10.1152/ajpendo.00239.2003
View details for Web of Science ID 000185822500005
View details for PubMedID 12876075
Inorganic polyphosphate (poly P), chains of hundreds of phosphate residues linked by "high-energy" bonds as in ATP, has been conserved from prebiotic times in all cells. Poly P is essential for a wide variety of functions in bacteria, including virulence in pathogens. In this study, we observe the unique and many-fold stimulation by poly P in vitro of the protein kinase mTOR (mammalian target of rapamycin). To explore the role of poly P in mammalian cells, a yeast polyphosphatase, PPX1, was inserted into the chromosomes of MCF-7 mammary cancer cells. The transfected cells are markedly deficient in their response to mitogens, such as insulin and amino acids, as seen in their failure to activate mTOR to phosphorylate one of its substrates, PHAS-I (the initiation factor 4E-binding protein). In addition, the transfected cells are severely reduced in their growth in a serum-free medium. On the basis of these findings, we suggest that poly P (and/or PPX1) serves as a regulatory factor in the activation of mTOR in the proliferative signaling pathways of animal cells.
View details for DOI 10.1073/pnas.1534805100
View details for Web of Science ID 000185685700016
View details for PubMedID 12970465
Prior studies had suggested that Akt activity is elevated in a subset of breast cancers. In this study, to test the effect of active Akt-3 on estrogen receptor function, we have produced MCF-7 cells, which express active Akt-3 and examined the estrogen responsiveness of these cells in vivo and in vitro. Experimental Design: MCF-7 cells expressing active Akt-3 were studied for estradiol (E2) responsiveness in vitro by both using an estrogen receptor element reporter construct as well as looking at induction of endogenous genes. These cells were also studied in vivo after injection into nude, ovariectomized mice by following tumor growth rates in the presence or absence of E2, tamoxifen, or the pure antiestrogen, ICI 182,780 (fulvestrant).Akt-3-expressing cells were found to produce tumors in mice in the absence of E2 that were approximately equivalent in size to control cells in mice given E2. Moreover, the formation of tumors by the Akt-3 cells was greatly suppressed by E2, stimulated by tamoxifen, and unaffected by ICI 182,780. In the in vitro assays for gene induction by E2, the Akt-3-expressing cells exhibited similar E2 and tamoxifen responsiveness as the control cells.These results indicate that expression of active Akt-3 in MCF-7 cells results in E2-independent tumor growth. Moreover, the growth of these tumors is inhibited by E2 and enhanced by tamoxifen. Finally, these tumors are resistant to ICI 182,780. These findings suggest that the amount of active Akt present in breast cancers may be important in the relative efficacy of different treatments.
View details for Web of Science ID 000184680200010
View details for PubMedID 12912939
To determine which genes may be regulated by Akt and participate in the transformation of cells, we have examined by microarray analyses genes turned on in the prostate cancer cell line, PC3, when Akt activity was induced. PC3 cells, which lack the lipid phosphatase PTEN, were treated overnight with a reversible inhibitor of the phosphatidylinositol 3-kinase, LY294002 (a treatment which was found to reversibly decrease Akt enzymatic activity). The inhibitor was then washed out and mRNA collected 2, 6, and 10 h later and compared by microarray analyses with mRNAs present immediately after removal of the inhibitor. One of the identified induced mRNAs, Fra-1, was further studied by transient transfections of a reporter construct containing its 5' regulatory region. This construct was found to be directly induced 4- to 5-fold by co-transfection with constitutively active Akt3 but not kinase dead Akt. The regulation by Akt3 was found to be due to two specific regions in the Fra-1 regulatory sequence which match Sp1 consensus sites. Finally, gel shift studies showed that the binding of Sp1 to one of these sites was dependent on the PI 3-kinase pathway. These results indicate that LY294002 treatment and washout is a useful method to study the activation of Akt in the context of a tumor cell. Moreover, the identification of Fra-1 as an Akt-regulated gene may have implications for the ability of Akt to transform cells since Fra-1 has been implicated in cell growth and the aggressiveness of tumors.
View details for Web of Science ID 000182325600008
View details for PubMedID 12692267
Akt (also called protein kinase B) is one of the major downstream targets of the phosphatidylinositol 3-kinase pathway. This protein kinase has been implicated in insulin signaling, stimulation of cellular growth, and inhibition of apoptosis as well as transformation of cells. Although a number of cellular proteins have been identified as putative targets of the enzyme, additional substrates may play a role in the varied responses elicited by this enzyme. We have used a combination of 14-3-3 binding and recognition by an antibody to the phosphorylation consensus of the enzyme to identify and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt was accomplished by showing that 1) PRAS40 was phosphorylated in vitro by purified Akt on the same site that was phosphorylated in insulin-treated cells; 2) activation of an inducible Akt was alone sufficient to stimulate the phosphorylation of PRAS40; and 3) cells lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate this protein. Thus, PRAS40 is a novel substrate of Akt, the phosphorylation of which leads to the binding of this protein to 14-3-3.
View details for DOI 10.1074/jbc.M210837200
View details for Web of Science ID 000181777500028
View details for PubMedID 12524439
Ser/Thr phosphorylation of insulin receptor substrate-1 (IRS-1) is a negative regulator of insulin signaling. One potential mechanism for this is that Ser/Thr phosphorylation decreases the ability of IRS-1 to be tyrosine-phosphorylated by the insulin receptor. An additional mechanism for modulating insulin signaling is via the down-regulation of IRS-1 protein levels. Insulin-induced degradation of IRS-1 has been well documented, both in cells as well as in patients with diabetes. Ser/Thr phosphorylation of IRS-1 correlates with IRS-1 degradation, yet the details of how this occurs are still unknown. In the present study we have examined the potential role of different signaling cascades in the insulin-induced degradation of IRS-1. First, we found that inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin block the degradation. Second, knockout cells lacking one of the key effectors of this cascade, the phosphoinositide-dependent kinase-1, were found to be deficient in the insulin-stimulated degradation of IRS-1. Conversely, overexpression of this enzyme potentiated insulin-stimulated IRS-1 degradation. Third, concurrent with the decrease in IRS-1 degradation, the inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin also blocked the insulin-stimulated increase in Ser(312) phosphorylation. Most important, an IRS-1 mutant in which Ser(312) was changed to alanine was found to be resistant to insulin-stimulated IRS-1 degradation. Finally, an inhibitor of c-Jun N-terminal kinase, SP600125, at 10 microm did not block IRS-1 degradation and IRS-1 Ser(312) phosphorylation yet completely blocked insulin-stimulated c-Jun phosphorylation. Further, insulin-stimulated c-Jun phosphorylation was not blocked by inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin, indicating that c-Jun N-terminal kinase is unlikely to be the kinase phosphorylating IRS-1 Ser(312) in response to insulin. In summary, our results indicate that the insulin-stimulated degradation of IRS-1 via the phosphatidylinositol 3-kinase pathway is in part dependent upon the Ser(312) phosphorylation of IRS-1.
View details for DOI 10.1074/jbc.M209153200
View details for Web of Science ID 000181466800059
View details for PubMedID 12510059
In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.
View details for Web of Science ID 000180715900003
View details for PubMedID 12374568
Estrogen acting through the estrogen receptor (ER) is able to regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors. Ligand-activated ER binds DNA and transactivates the promoters of estrogen target genes. In addition, ligand-activated ER can interact with other factors to alter the physiology and growth of cells. Using a yeast two-hybrid screen, we have identified an interaction between ER alpha and the proapoptotic forkhead transcription factor FKHR. The ER alpha-FKHR interaction depends on beta-estradiol and is reduced significantly in the absence of hormone or the presence of Tamoxifen. A glutathione S-transferase pull-down assay was used to confirm the interaction and localized two interaction sites, one in the forkhead domain and a second in the carboxyl terminus. The FKHR interaction was specific to ER alpha and was not detected with other ligand-activated steroid receptors. The related family members, FKHRL1 and AFX, also bound to ER alpha in the presence of beta-estradiol. FKHR augmented ER alpha transactivation through an estrogen response element. Conversely, ER alpha repressed FKHR-mediated transactivation through an insulin response sequence, and cell cycle arrest induced by FKHRL1 in MCF7 cells was abrogated by estradiol. These results suggest a novel mechanism of estrogen action that involves regulation of the proapoptotic forkhead transcription factors.
View details for Web of Science ID 000170910200037
View details for PubMedID 11435445
The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.
View details for Web of Science ID 000167474900057
View details for PubMedID 11145958
In the present studies, we demonstrate that heregulin is a potent and rapid activator of the serine/threonine kinase called Akt in the MCF-7 breast cancer cell line but not in 3 other breast cancer cell lines (T47D, HBL-100, and MDA-231). The extent of activation of Akt in the 4 cell lines correlated with the ability of heregulin to activate phosphatidylinositol 3-kinase and inhibition of the kinase blocked Akt activation. A monoclonal antibody to HER2 inhibited the ability of heregulin to activate Akt in the MCF-7 cells. BT474, a breast cancer cell line which overexpresses HER2, had high basal Akt enzymatic activity. This high basal activity was lowered when cells were pre-incubated with an anti-HER2 monoclonal antibody which is used to treat breast cancer patients. Our results indicate that heregulin is a potent activator of Akt and that overexpression of HER2 in breast cancers could also lead to activation of Akt.
View details for Web of Science ID 000082014900060
View details for PubMedID 10441522
We measured the insulin-stimulated amount of Akt1, Akt2, and Akt3 enzymatic activities in four breast cancer cell lines and three prostate cancer cell lines. In the estrogen receptor-deficient breast cancer cells and the androgen-insensitive prostate cells, the amount of Akt3 enzymatic activity was approximately 20-60-fold higher than in the cells that were estrogen- or androgen-responsive. In contrast, the levels of Akt1 and -2 were not increased in these cells. The increase in Akt3 enzyme activity correlated with an increase in both Akt3 mRNA and protein. In a prostate cancer cell line lacking the tumor suppressor PTEN (a lipid and protein phosphatase), the basal enzymatic activity of Akt3 was constitutively elevated and represented the major active Akt in these cells. Finally, reverse transcription-PCR was used to examine the Akt3 expression in 27 primary breast carcinomas. The expression levels of Akt3 were significantly higher in the estrogen receptor-negative tumors in comparison to the estrogen receptor-positive tumors. To see if the increase in Akt3 could be due to chromosomal abnormalities, the Akt3 gene was assigned to human chromosome 1q44 by fluorescence in situ hybridization and radiation hybrid cell panel analyses. These results indicate that Akt3 may contribute to the more aggressive clinical phenotype of the estrogen receptor-negative breast cancers and androgen-insensitive prostate carcinomas.
View details for Web of Science ID 000081721100009
View details for PubMedID 10419456
We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.
View details for Web of Science ID 000081438300040
View details for PubMedID 10400647
The family of protein kinases called Akt, protein kinase B (PKB), or related to A and C kinase (RAC) have been implicated in numerous biological processes including adipocyte and muscle differentiation, glycogen synthesis, glucose uptake, apoptosis and cellular proliferation. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1 and 2 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. However, the reported sequence of the rat Akt3 protein differed significantly from this in that it lacked 25 amino acids in the C-terminal region, including this key regulatory serine phosphorylation site (Biochem. Biophys. Res. Commun. 216, 526-534). In the present studies we show that the deduced sequence of human Akt3 contains this serine and that it is phosphorylated in response to insulin. These results indicate that human Akt3 is regulated similarly to Akt1 and Akt2.
View details for Web of Science ID 000079925300047
View details for PubMedID 10208883
Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF did not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) were required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt was found to inhibit insulin signaling. Moreover, this inhibition required the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt were inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade.
View details for Web of Science ID 000079451800032
View details for PubMedID 10092613
A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMP-stimulated increase in PEPCK gene transcription.
View details for Web of Science ID 000076448000045
View details for PubMedID 9765258
In the present work a chimeric receptor containing the intracellular domain of the insulin receptor-related receptor (IRR) and the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor was expressed in 3T3-L1 adipocytes and compared with the parallel chimeric receptor containing the cytoplasmic domain of the insulin receptor (IR). Both chimeric receptors exhibited CSF-stimulated tyrosine kinase activity when assayed in vitro after in vivo activation comparable to that of the endogenous IR present in these cells. No cross-activation of the expressed chimeric and endogenous receptors was observed. The cytoplasmic domain of the IRR was found to 1) mediate activation of the Ser/Thr kinase Akt/PKB, 2) stimulate glucose uptake, 3) inhibit lipolysis, and 4) stimulate glycogen synthase, all with a potency comparable to those of the expressed CSF-1R/IR chimera and the endogenous insulin receptors. These results indicate that despite the extensive differences in sequence between the cytoplasmic domains of the IRR and IR, the elements required for insulin-specific responses have been conserved in this distinct member of the insulin receptor family.
View details for Web of Science ID 000074922600027
View details for PubMedID 9681510
The activation of protein kinase B/Akt is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. Because insulin-like growth factor 1 stimulates the resumption of meiosis in Xenopus laevis oocytes via phosphoinositide 3-kinase activation, we investigated the Akt involvement in this process. Injection of mRNA coding for a constitutively active Akt in Xenopus oocytes induced germinal vesicle breakdown (GVBD) to the same extent as progesterone or insulin treatment. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a lysine mutation in the catalytic domain that abolishes the kinase activity had no effect. A mutant Akt lacking a membrane-targeting sequence did not induce GVBD, despite high levels of expression and activity. As previously reported for insulin, induction of GVBD by Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for the type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE is required for Akt action. That an increase in PDE activity in the oocyte is sufficient to induce meiotic resumption was demonstrated by expression of an active PDE protein. In addition, the constitutively active Akt caused a 2-fold increase in the activity of the endogenous PDE. These data demonstrate that Akt is in the pathway controlling resumption of meiosis in the Xenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation.
View details for Web of Science ID 000074974700008
View details for PubMedID 9668041
Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.
View details for Web of Science ID 000073536700076
View details for PubMedID 9565622
In the present studies, we have compared the properties of two members of the Akt family of ser/thr kinases, Akt1 and Akt3. First, we demonstrate that both 3T3-L1 fibroblasts and adipocytes express Akt3 mRNA by RT-PCR and sequencing of the resultant PCR product. Second, we show that insulin stimulates the enzymatic activity of Akt1 and Akt3 15- and 7-fold, respectively. We then investigated the ability of protein kinase C to regulate Akt1 and 3. Neither enzyme was activated by stimulation of protein kinase C, however, the insulin-stimulated increases in activity of both isozymes were found to be comparably inhibited by prior protein kinase C activation. Since this inhibition could have resulted from an interaction of the pleckstrin homology domain of the Akt with protein kinase C, we also examined the ability of a mutant Akt1 lacking this domain to be regulated by this enzyme. The insulin-stimulated increase in enzymatic activity of this mutant Akt was regulated by PKC activation like the wild type enzyme. These results indicate that Akt1 and 3 are similarly stimulated by insulin and this stimulation is inhibited by prior activation of protein kinase C through a mechanism that is independent of the presence of the pleckstrin homology domain.
View details for Web of Science ID 000072130300031
View details for PubMedID 9480839
The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.
View details for Web of Science ID 000071325200001
View details for PubMedID 9443070
Increased serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been observed in several systems to correlate with a decreased ability of the insulin receptor to tyrosine-phosphorylate this endogenous substrate and to inhibit its subsequent association with phosphatidylinositol 3-kinase. In the present studies we have examined the potential role of the mitogen-activated protein (MAP) kinase in the increased serine phosphorylation of IRS-1 observed in human embryonic kidney cells treated with an activator of protein kinase C, phorbol 12-myristate 13-acetate. First, recombinantly produced kinase was shown to phosphorylate intact IRS-1 in a way that decreased the ability of isolated insulin receptor to phosphorylate the tyrosines recognized by the SH2 domains of the phosphatidylinositol 3-kinase. Second, an inhibitor of MAP kinase activation, PD98059, blocked the phorbol 12-myristate 13-acetate-induced inhibition of the insulin-stimulated increase in IRS-1 associated phosphatidylinositol 3-kinase. Third, activation of MAP kinase in intact cells via a regulatable upstream kinase, a RAF:estrogen receptor construct, could also inhibit the insulin-stimulated increase in IRS-1-associated phosphatidylinositol 3-kinase. Fourth, an in gel kinase assay showed that MAP kinase was the primary renaturable kinase in cell extracts capable of phosphorylating an IRS-1 fusion protein. Finally, IRS-1 was found to associate in coprecipitation studies with endogenous MAP kinase. These studies implicate MAP kinase as one of the kinases capable of phosphorylating and regulating IRS-1 tyrosine phosphorylation.
View details for Web of Science ID A1997YL41900030
View details for PubMedID 9395471
Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.
View details for Web of Science ID A1997YD31500033
View details for PubMedID 9335553
The expression of the ob gene product leptin in adipose tissues has been previously described to be regulated by insulin in vivo and vitro. Akt, a ser/thr kinase with a pleckstrin homology domain, has recently been identified to function in the insulin receptor signaling cascade. The aim of this study was to investigate the role of Akt in the production of leptin by adipocytes. Therefore, we examined leptin production by 3T3-L1 adipocytes stably expressing a myristoylated version of Akt which is constitutively active. Leptin levels in the supernatants of serum starved, nonstimulated 3T3-L1 adipocytes were determined by radioimmunoassay (RIA). Expression of the constitutively active Akt was found to induce a more than 20-fold increase in leptin levels whereas a control non-myristoylated Akt had no effect. Leptin mRNA levels as determined by either RNase protection assay or reverse transcriptase (RT)-polymerase chain reaction (PCR) were not elevated by the constitutively active Akt. These results indicate that Akt can induce leptin production in 3T3-L1 adipocytes via a non-transcriptional mechanism.
View details for Web of Science ID A1997XL84200060
View details for PubMedID 9231812
Prior studies have shown that Madin-Darby canine kidney cells (MDCK) overexpressing the human insulin receptor bind and respond normally to insulin (T.C. Yeh, R.A. Roth, Diabetes 43 (1994) 1297-1303). Moreover, the insulin receptor preferentially localizes to the basolateral membrane of these cells. In the present studies, insulin was added to either the apical or the basolateral side of these cells and the extent of degradation of the insulin was assessed. Radioactive insulin added to either side was bound to its receptor and the radioactivity which reached the other side of the cell was to a large extent degraded fragments. Insulin added to the apical side was degraded to a larger extent (83%) than when added to the basolateral side (49%) although the basolateral side has much more insulin receptors than the apical side. This degradation process was not inhibitors of either lysosomal enzymes, the proteasome complex or cathepsins. The degradation process could however, be potently inhibited by the sulfhydryl alkylating agent N-ethylmaleimide. Further, cell surface biotinylation study showed that the insulin degrading enzyme was preferentially localized on the apical membranes. These results suggest that insulin added on the apical side of MDCK cells are more closely linked to the degradation process than that added on the basolateral side.
View details for Web of Science ID A1997XQ99000001
View details for PubMedID 9279478
To investigate the role of insulin degrading enzyme (insulysin, EC 184.108.40.206) in insulin signaling, Chinese hamster ovary cells overexpressing the human insulin receptor were genetically engineered to also stably overexpress the rat insulin degrading enzyme. In comparison to the parental cells, these cells expressed 2.7-fold elevated levels of enzyme and insulin degradation was also increased 2-fold. These cells also exhibited a more rapid decrease in receptor tyrosine phosphorylation after removal of insulin. Moreover, low concentrations of insulin were less effective at stimulating proliferation of the cells overexpressing the enzyme. Finally, a fraction of the overexpressed enzyme as well a fraction of the endogenous enzyme could be detected on the plasma membrane surface of these cells. These results support the hypothesis that this enzyme may function in insulin signaling by degrading the insulin molecule.
View details for Web of Science ID A1997WH35500036
View details for PubMedID 9070242
Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present studies, the constitutively active Akt and a nonmyristoylated control mutant were expressed in 3T3-L1 cells that can be induced to differentiate into adipocytes. The constitutively active Akt induced glucose uptake into adipocytes in the absence of insulin by stimulating translocation of the insulin-responsive glucose transporter 4 to the plasma membrane. The constitutively active Akt also increased the synthesis of the ubiquitously expressed glucose transporter 1. The increased glucose influx in the 3T3-L1 adipocytes directed lipid but not glycogen synthesis. These results indicate that Akt can regulate glucose uptake and metabolism.
View details for Web of Science ID A1996VW68600056
View details for PubMedID 8940145
Prior studies have demonstrated that a juxtamembrane tyrosine (tyrosine 972) in the insulin receptor is required for the receptor to elicit various biological responses and to stimulate the tyrosine phosphorylation of two endogenous substrates, the insulin receptor substrate-1 and the adaptor protein called Shc. In the present studies the role of this tyrosine was examined in the insulin-stimulated tyrosine phosphorylation of a group of 60-kDa endogenous proteins. These include a 60-kDa protein which, when phosphorylated, becomes associated with the GTPase activating protein of Ras, a distinct 60-kDa protein that associates with either the phosphatidylinositol 3-kinase or the tyrosine phosphatase Syp, as well as a 58/53-kDa protein that is tyrosine phosphorylated in response to insulin but has no known associated protein. In each case, a mutant insulin receptor in which tyrosine 972 has been changed to phenylalanine was found to be defective in its ability to phosphorylate these three endogenous substrates, although the mutant receptor exhibited the same level of insulin-stimulated autophosphorylation as the wild type receptor. These results further demonstrate the critical role that the juxtamembrane tyrosine 972 plays in downstream signaling by the insulin receptor.
View details for Web of Science ID A1996VU35100021
View details for PubMedID 8940353
Akt is a serine/threonine kinase that is stimulated by receptor tyrosine kinases and contains a pleckstrin homology domain. One model proposed to explain this activation suggests that receptor tyrosine kinases stimulate a phosphatidylinositol 3-kinase whose lipid products directly activate Akt kinase by interacting with its pleckstrin homology domain. In the present study, we show, in three cell types, that Akt does not require its pleckstrin homology domain to respond to either insulin or platelet-derived growth factor. Moreover, attachment of the src myristoylation signal to target Akt, without its pleckstrin homology domain, to the membrane constitutively activates Akt by causing an increase in its basal level of phosphorylation. This constitutively active form of Akt can also activate p70(S6K), indicating that the pleckstrin homology domain is not necessary for downstream interactions. Fusion of the inter src homology 2 domain from the p85 regulatory subunit of the phosphatidylinositol 3-kinase to Akt also constitutively activated Akt and induced an association with the lipid kinase. Phosphorylation of this fusion protein still critically contributes toward its increased activity. The sum of these results indicates that the primary mechanism of Akt activation is via protein phosphorylation.
View details for Web of Science ID A1996VF61200037
View details for PubMedID 8702995
A monoclonal antibody has been produced which immunoprecipitates 58- and 53-kDa proteins which are rapidly tyrosine phosphorylated in insulin-treated cells. These proteins can also be tyrosine phosphorylated in vitro by the isolated human insulin receptor. Increased tyrosine phosphorylation of these proteins is also observed in cells expressing a transforming chicken c-Src (mutant Phe-527) and in cells with the activated tyrosine kinase domains of the Drosophila insulin receptor, human insulin-like growth factor I receptor, and human insulin receptor-related receptor. P58/53 did not appear to associate with either the GTPase activating protein of Ras (called GAP) or the phosphatidylinositol 3-kinase by either co-immunoprecipitation experiments or in Far Westerns with the SH2 domains of these two proteins. Since p58/53 did not appear, by immunoblotting, to be related to any previously described tyrosine kinase substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase Syp, the protein was purified in sufficient amounts to obtain peptide sequence. This sequence was utilized to isolate a cDNA clone that encodes a previously uncharacterized 53-kDa protein which, when expressed in mammalian cells, is tyrosine phosphorylated by the insulin receptor.
View details for Web of Science ID A1996TU69100010
View details for PubMedID 8621681
In the present study we describe a nonradioactive assay for measuring the intrinsic tyrosine kinase activity of the insulin receptor. This assay utilizes as an exogenous substrate a biotinylated peptide based on the sequence of the endogenous substrate insulin receptor substrate-1 (IRS-1). To separate the tyrosine phosphorylated peptide from the nonphosphorylated peptide, immobilized recombinantly produced SH2 domain of the p85 subunit of the phosphatidylinositol 3-kinase is utilized to bind the tyrosine-phosphorylated peptide. The amount of bound peptide is then detected by the use of peroxidase-conjugated streptavidin and a colorimetric assay. This assay has been used to measure the tyrosine kinase activity of receptor which was immunocaptured from lysates of various cells overexpressing the human insulin receptor as well as the endogenous insulin receptors in the parental cells. In this in vitro assay, no decrease in tyrosine kinase activity was observed in receptors from cells with activated overexpressed protein kinase C alpha or after high glucose treatment although a decrease in in situ phosphorylation of IRS-1 was observed with the activation of protein kinase C alpha. These results indicate that this assay may be a useful new method for monitoring the enzymatic activity of the insulin receptor kinase as well as other tyrosine kinases.
View details for Web of Science ID A1995TJ97900008
View details for PubMedID 8594980
To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.
View details for Web of Science ID A1995TB46700072
View details for PubMedID 7479769
Stimulation of the activity of protein kinase C by pretreatment of cells with phorbol esters was tested for its ability to inhibit signaling by four members of the insulin receptor family, including the human insulin and insulin-like growth factor-I receptors, the human insulin receptor-related receptor, and the Drosophila insulin receptor. Activation of overexpressed protein kinase C alpha resulted in a subsequent inhibition of the ligand-stimulated increase in antiphosphotyrosine-precipitable phosphatidylinositol 3-kinase mediated by the kinase domains of all four receptors. This inhibition varied from 97% for the insulin receptor-related receptor to 65% for the Drosophila insulin receptor. In addition, the activation of protein kinase C alpha inhibited the in situ ligand-stimulated increase in tyrosine phosphorylation of the GTPase-activating protein-associated p60 protein as well as Shc mediated by these receptors. The mechanism for this inhibition was further studied in the case of the insulin-like growth factor-I receptor. Although the in situ phosphorylation of insulin-receptor substrate-1 and p60 by this receptor was inhibited by prior stimulation of protein kinase C alpha, the in vitro tyrosine phosphorylation of these two substrates by this receptor was not decreased by prior stimulation of the protein kinase C alpha in the cells that served as a source of the substrates. Finally, the insulin-like growth factor-I-stimulated increase in cell proliferation was found to be inhibited by prior activation of protein kinase C alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1995RU75700029
View details for PubMedID 7545165
In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
View details for Web of Science ID A1995RU75100018
View details for PubMedID 7556070
Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the drosophila insulin receptor (IR) with the extracellular domain of the human IR were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bound insulin equally and exhibited an insulin-activated tyrosine kinase activity. The ability of the drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase was found to be indistinguishable from that of the human IR. The chimeric drosophila receptors did not bind more phosphatidylinositol 3-kinase than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors.
View details for Web of Science ID A1995QU26000007
View details for PubMedID 7711018
In the present studies, insulin was found to stimulate in a rat hepatoma cell line (called FAO cells) the tyrosine phosphorylation of the 60-kilodalton p21ras GTPase-activating protein (GAP)-associated protein called p60. Surprisingly, the tyrosine phosphorylation of this protein was also almost equally stimulated by an activator of protein kinase C (PKC), the phorbol ester phorbol 12-myristate 13-acetate (PMA). The tyrosine phosphorylation of p60 induced by either agent correlated with the formation of the GAP-p60 complex in situ and an increase in the ability of p60 to directly bind to the SH2 domain of GAP in vitro. Several lines of evidence indicated that the PMA-induced tyrosine phosphorylation of p60 occurred through a different mechanism than that induced by insulin. First, the stimulation of tyrosine phosphorylation of p60 by maximal concentrations of the two agents was almost additive. Second, down-regulation of PKC or pretreatment with a specific inhibitor of PKC abolished the ability of PMA to stimulate tyrosine phosphorylation of p60 but had no effect on the insulin stimulation. And third, long-term pretreatment with insulin abolished the insulin response but did not affect the response to PMA. The PMA effect did seem to be mediated via a tyrosine kinase, since it was blocked by quercetin, an inhibitor of tyrosine kinases. These results indicate that both PMA and insulin can equally stimulate in FAO cells the tyrosine phosphorylation of p60 and its association with GAP, although these two agents seem to act via different signaling systems.
View details for Web of Science ID A1995QD58600014
View details for PubMedID 7835279
A gene encoding a putative third member of the insulin receptor family (called the insulin receptor-related receptor or IRR) was isolated in 1989. However, the naturally occurring protein product encoded by this gene has yet to be described. In the present studies, we have generated four monoclonal antibodies to a recombinantly expressed chimera, which contains the extracellular domain of human IRR. These antibodies were found to specifically recognize the chimeric IRR (and not the insulin or insulin-like growth factor I receptors), and two of the antibodies were capable of acting as partial agonists in the cells expressing the chimeric IRR. These antibodies have therefore been utilized to study the expression and properties of the native receptor. In contrast to the two other members of this receptor family, the endogenous IRR protein had only a very limited expression, being detected only in neuroblastomas. In primary neuroblastomas, the levels of the receptor were highest in samples from stage A tumors (those which are generally more highly differentiated and have higher levels of the nerve growth factor receptor). The endogenous IRR could also be detected in a neuroblastoma cell line (called IMR-5 cells). In these cells, IRR could be shown to be partly present as a hybrid with the insulin and insulin-like growth factor-I receptors but not with the receptor for nerve growth factor. The intrinsic tyrosine kinase activity of this endogenous IRR was activated by the agonist monoclonal antibody to IRR but not by nerve growth factor, insulin-like growth factor I, or insulin. Finally, this monoclonal antibody was found to stimulate mitogen-activated protein kinase activity in these cells. In summary, these studies demonstrate for the first time that the IRR protein is normally expressed, that its levels are highest in neuronal tissues, and that it can form hybrid receptors with the two other members of this receptor family but not with the more distantly related nerve growth factor receptor.
View details for Web of Science ID A1995QD20400060
View details for PubMedID 7829525
Protein(s) which bind polyphosphatidylinositol phosphates (PI 3,4,5-P3 and PI 4,5-P2) were identified in the wheat-germ agglutinin bound fraction of cells and tissues. The binding of this protein(s) to the phospholipid could be demonstrated in two ways, either by a shift in the migration of the lipid by size exclusion column chromatography or directly by binding to the protein after capture on wheat-germ agglutinin-coupled beads. Of the rat tissues tested (muscle, spleen, brain, heart, kidney and liver), the activity was highest in liver. The protein(s) was purified more than 5000-fold by sequential chromatography on columns of wheat-germ agglutinin, phosphocellulose, Blue-Sepharose, Mono Q and Superose 6. The peak of activity appeared to have a molecular weight on this latter column of approx. 240,000. The protein(s) bound PI 3,4,5-P3, PI 3,4-P2, and PI 3-P in the ratio of 4:2:1. The binding of 3-phosphorylated PI phosphates to the protein(s) was not significantly inhibited by 36 micrograms/ml of either phosphatidylinositol or phosphatidylcholine, but was inhibited 10% and 65% by 36 micrograms/ml of PI 4-P and PI 4,5-P2, respectively. Since these results suggested that the binding protein(s) could also bind PI 4,5-P2, binding of this lipid was directly tested and found to be comparable to that of PI 3,4,5-P3. These results suggest that this protein(s) could be involved in the signaling mechanism elicited by these polyphosphoinositides.
View details for Web of Science ID A1994QA37700029
View details for PubMedID 7803513
A monoclonal antibody to a 60-kDa substrate of the insulin receptor tyrosine kinase is utilized in the present studies to examine this molecule in 3T3 cells expressing either the transforming chicken c-Src (mutant Phe-527), the wild type molecule, or the parental cells. The tyrosine phosphorylation of this 60-kDa protein was greatly increased in cells expressing transforming Src and partially increased in cells expressing wild type enzyme. This tyrosine phosphorylation correlated with an increased association with the GTPase-activating protein of p21ras (GAP). However, this 60-kDa protein did not react with antibodies to another 62-kDa tyrosine-phosphorylated protein previously isolated from Src-transformed cells (Wong, G., Muller, O., Clark, R., Conroy, L., Moran, M. F., Polakis, P., and McCormick, F. (1992) Cell 69, 551-558), although this latter antibody did react with a 62-kDa protein in anti-phosphotyrosine precipitates from cells expressing transforming c-Src but not the parental cells. These two proteins could also be distinguished by their subcellular location, the ability of the latter but not the former protein to bind RNA, and their migration in SDS gels. Moreover, the 62-kDa RNA-binding phosphoprotein could be almost completely depleted from cell lysates with poly(U)-Sepharose without affecting the amount of either the GAP-associated 60-kDa tyrosine-phosphorylated protein or the protein precipitated with the monoclonal antibody. When the two proteins were phosphorylated in vitro with purified c-Src, they were both found to bind directly to the amino-terminal SH2 domain of GAP, although the RNA-binding protein was found to have a weaker affinity. These results indicate that two distinct 60-kDa proteins are substrates for the Src tyrosine kinase, one which binds RNA and the other which constitutes the major GAP-associated 60-kDa phosphoprotein.
View details for Web of Science ID A1994PU28400047
View details for PubMedID 7525585
We have developed and characterized a line of Madin-Darby canine kidney (MDCK) cells overexpressing the human insulin receptor. The expressed receptor was found to be processed normally, and its intrinsic tyrosine kinase was determined to be functional from both in vitro and in vivo phosphorylation studies. The expressed receptor was able to mediate an insulin-stimulated increase in both anti-phosphotyrosine-precipitable and anti-insulin receptor substrate 1-precipitable phosphatidylinositol 3-kinase activity. Moreover, insulin-induced glycogen synthase activity was greater and more sensitive to insulin in the transfected cells than in the parental cells. Interestingly, insulin promoted tubule-like growth in cells overexpressing the insulin receptor but not in the parental cells. Another advantage of this cell system lies in its ability to polarize into distinct basolateral and apical membrane compartments. With the use of biotinylation and Western analysis, the expressed insulin receptor was found to be preferentially expressed in the basolateral membrane (fivefold greater) in comparison with the apical membrane. Therefore, MDCK cells overexpressing the insulin receptor represent a novel system to study not only the pathway of insulin signaling, but also this pathway in the context of cell polarity.
View details for Web of Science ID A1994PN28100005
View details for PubMedID 7926303
Maturation of the insulin proreceptor in a late Golgi compartment requires cleavage at an Arg-Lys-Arg-Arg processing site, suggesting involvement of furin, a transmembrane serine protease of the Kex2 family of processing enzymes. A genetically engineered secreted, soluble form of human furin (ss-furin), expressed by infection of insect cells with a recombinant baculovirus, was purified to near homogeneity. ss-Furin exhibited rapid and efficient cleavage of both isoforms of the human insulin proreceptor in solubilized extracts of cultured mammalian cells expressing preproreceptor cDNA. Proreceptor cleavage occurred at the physiological processing site as judged by the effects of mutations in this site on cleavage by purified ss-furin. Moreover, purified ss-furin exhibited specificity for proreceptor cleavage identical to that of the endogenous insulin proreceptor-processing enzyme. Furin thus displays the properties expected of an insulin proreceptor-processing enzyme in that it (i) cleaves the proreceptor efficiently and at the correct site; (ii) exhibits the same specificity in processing variant proreceptors as the endogenous enzyme; (iii) appears to be localized in the correct secretory compartment; and (iv) has the same broad pattern of tissue distribution as the insulin proreceptor.
View details for Web of Science ID A1994PQ49100088
View details for PubMedID 7929288
A new site of serine phosphorylation (Ser-1035/1037) has been identified in the kinase domain of the insulin receptor. Mutant receptors missing these two serines were expressed in Chinese hamster ovary cells overexpressing protein kinase C alpha. These mutant receptors lacked a phorbol ester-stimulated phosphoserine containing tryptic peptide as demonstrated by both high percentage polyacrylamide/urea gel electrophoresis and two-dimensional tlc. Moreover, a synthetic peptide with the sequence of this tryptic peptide was phosphorylated by isolated protein kinase C alpha and co-migrated with the phosphopeptide from in vivo labeled receptor. These results indicate that serine-1035 and/or 1037 in the kinase domain of the insulin receptor are phosphorylated in response to activation of protein kinase C alpha.
View details for Web of Science ID A1994PK72700029
View details for PubMedID 7926007
Insulin, in the presence of phorbol esters, was observed to stimulate the tyrosine phosphorylation of a major 80 kDa protein by immunoblotting with anti-phosphotyrosine antibodies in Chinese hamster ovary cells overexpressing the insulin receptor and protein kinase C alpha. The protein was specifically immunoprecipitated by antibodies to protein kinase C and anti-phosphotyrosine antibodies were capable of immunoprecipitating protein kinase C enzymatic activity from these cells. When this tyrosine phosphorylated protein kinase C was treated with a tyrosine-specific phosphatase, a 35% decrease in its enzymatic activity was observed and this inhibition was blocked by inclusion of a tyrosine phosphatase inhibitor, vanadate, in the reaction mixture. These results indicate that under certain conditions insulin can stimulate the tyrosine phosphorylation of protein kinase C and this phosphorylation can affect its enzymatic activity.
View details for Web of Science ID A1994NL38800057
View details for PubMedID 7514404
A line of Chinese hamster ovary cells overexpressing protein kinase C alpha was transfected with cDNAs encoding either the wild-type human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.
View details for Web of Science ID A1994NA07300033
View details for PubMedID 8135757
View details for PubMedID 8205066
Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding protein kinase C alpha (PKC) and a cell line (CHO-PKC alpha) expressing approximately 7-fold greater amounts of PKC as the parental cells were isolated. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate in the CHO-PKC alpha cells inhibited by approximately 75% the: 1) insulin-stimulated increase in antiphosphotyrosine precipitable phosphatidylinositol 3-kinase activity in these cells; 2) insulin-stimulated increase in PI 3-kinase activity associated with insulin receptor substrate-1; and 3) tyrosine phosphorylation of the endogenous substrate, insulin receptor substrate-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate treatment did not inhibit any of these responses in the parental CHO cells. These results indicate that excessive PKC activity can interfere in a very early step in insulin receptor signaling and are consistent with the hypothesis that excessive PKC activity may contribute to some states of insulin resistance.
View details for Web of Science ID A1994MT06400006
View details for PubMedID 7512195
A novel active site has been identified in a family of zinc-dependent metalloendopeptidases that includes bacterial proteinase III, the human and Drosophila insulin-degrading enzymes, and the processing-enhancing protein subunit of the mitochondrial processing proteinase. None of these enzymes contains the conserved active site described in most other metalloendopeptidases, HEXXH; instead, all four contain an inversion of this motif, HXXEH. Prior mutagenesis studies of proteinase III indicate that the two histidines are essential for co-ordinating the zinc atom, while all three residues are required for enzyme activity. To identify the third zinc-binding residue in this protein family, three glutamates downstream from the active site were mutated to glutamine in proteinase III. The mutant proteins were expressed and their ability to degrade insulin was compared with the wild-type enzyme. The glutamate-204 mutant was as active as the wild-type protein, the glutamate-162 mutant retained 20% of the activity of the wild-type enzyme and the glutamate-169 mutant was completely devoid of insulin-degrading activity. The purified wild-type and glutamate-204 mutant enzymes were found to contain nearly stoichiometric levels of zinc by atomic absorption spectrophotometry, whereas the glutamate-162 mutant had a slight reduction in the level of zinc, and the glutamate-169 mutant retained less than 0.3 mol of zinc/mol of enzyme. These findings are consistent with glutamate-169 being the third zinc-binding residue in proteinase III.
View details for Web of Science ID A1993LD11300021
View details for PubMedID 8099278
Insulin stimulated tyrosine phosphorylation of SHC, a SH2 containing protein, was demonstrated in Chinese hamster ovary cells overexpressing the insulin receptor by immunoblotting with antiphosphotyrosine antibodies and in vivo labeling. Insulin induced tyrosine phosphorylation of SHC occurred very rapidly (within 1 min) with a dose curve which paralleled the autophosphorylation of the insulin receptor. Phosphorylation of SHC appeared to occur to a high stoichiometry since insulin induced the majority of SHC to shift to a higher molecular weight. The tyrosine phosphorylated SHC was not bound by the GTPase activating protein of Ras although a distinct 62 kDa tyrosine phosphorylated protein was found to be associated in the same experiments. It also was not bound to the insulin receptor, phosphatidylinositol 3-kinase or insulin receptor substrate-1.
View details for Web of Science ID A1993LC53100046
View details for PubMedID 7685165
Chinese hamster ovary cells overexpressing the human insulin receptor were transfected with cDNAs encoding protein kinase C isoenzymes alpha, beta I, gamma, and epsilon as well as an inactive alpha. Overexpression of these protein kinase Cs did not affect expression of the insulin receptor or insulin-stimulated tyrosine phosphorylation of the receptor. However, in response to phorbol esters, cells overexpressing isoenzymes alpha, beta I, and gamma, but not epsilon or inactive alpha, exhibited 3-4-fold higher levels of insulin receptor phosphorylation. This increased phosphorylation occurred exclusively on serines and threonine. Tryptic peptide maps indicated that this phosphorylation was primarily on serines 1305/1306 and threonine 1348 as well as several other unidentified sites. This phorbol ester-stimulated phosphorylation did not inhibit activation of the insulin receptor kinase when the receptor was activated in situ but assayed in vitro. However, in cells overexpressing protein kinase C alpha, it did inhibit an in vivo monitor of the activation of the insulin receptor kinase, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase activity. These results indicate that increased protein kinase C alpha activity can inhibit insulin-stimulated responses and support the hypothesis that excessive protein kinase C is involved in the insulin resistance observed in non-insulin-dependent diabetics.
View details for Web of Science ID A1993KT36800041
View details for PubMedID 8454604
In 1989, Shier and Watt identified a gene which was predicted to encode a new member of the insulin receptor (IR) family, and they called it the insulin receptor-related receptor (IRR) (Shier, P., and Watt, V. M. (1989) J. Biol. Chem. 264, 14605-14608). However, the tissues expressing this receptor, its ligand binding specificity and its signaling capability have remained unknown. In the present studies we report Northern blot analyses and polymerase chain reaction data, which indicate that the IRR mRNA is expressed in a variety of tissues, including the human kidney, heart, skeletal muscle, liver, and pancreas. In order to examine the ligand(s) recognized by IRR, we constructed a chimeric receptor with the extracellular domain of the IR replaced with that of IRR. This chimera was found not to bind radioactively labeled insulin, insulin-like growth factor I (IGF-I), or IGF-II. These ligands and relaxin, the only other known member of the mammalian insulin family, also failed to stimulate the tyrosine kinase activity of this chimeric receptor. A second chimeric receptor with the extracellular domain of IR and the kinase domain of IRR was also constructed and utilized to study the signaling capabilities of the kinase domain of IRR. This chimera exhibited high affinity insulin binding and insulin-stimulated tyrosine kinase activity. The kinase domains of the IR and IRR were found capable of phosphorylating the same spectrum of exogenous and endogenous substrates. However, Chinese hamster ovary (CHO) cells stably overexpressing the kinase domain of IRR exhibited elevated basal thymidine incorporation and 2-deoxyglucose uptake compared with CHO cells and CHO cells overexpressing wild-type IR. We conclude that: 1) IRR is expressed in the human kidney, heart, skeletal muscle, liver, and pancreas, 2) IRR does not appear to be the receptor of any known member of the insulin family, and 3) the tyrosine kinase of IRR appears to be similar to that of IR in both the spectrum of substrates phosphorylated and the biological responses stimulated.
View details for Web of Science ID A1992JN50200015
View details for PubMedID 1326521
An unusual active site has been identified in a family of zinc metalloendopeptidases that includes bacterial protease III and the human and Drosophila insulin-degrading enzymes. All of these enzymes have been characterized as metalloendopeptidases and purified protease III has been shown to contain stoichiometric levels of zinc. However, all three proteases lack the consensus sequence (HEXXH) described in the active site of other zinc metalloendopeptidases. Instead, these proteases contain an inversion of this motif, HXXEH. To determine whether this region could represent the active site in these proteins, the two histidines in protease III were individually mutated to arginine and the glutamate was mutated to glutamine. All three mutants were devoid of proteolytic activity toward an exogenous substrate, insulin, as compared to the wild-type protease. Three lines of evidence indicate that this loss of activity in the mutants is not due to distortion of the three-dimensional structure of the protein: (i) the mutants are secreted into the periplasmic space and chromatograph normally; (ii) all three mutants are expressed at levels nearly identical to wild-type protein and do not appear to have an increased susceptibility to proteolysis in the bacteria; and (iii) the mutants compete equally with wild-type protein in a radioimmunoassay. The purified wild-type and glutamate mutants were found to contain stoichiometric amounts of zinc by atomic absorption spectrophotometry, whereas both histidine mutants had negligible zinc signals. These findings are consistent with this region being the active site in this protein, with the histidine residues coordinating the essential zinc atom and the glutamate involved in catalysis.
View details for Web of Science ID A1992HR85300034
View details for PubMedID 1570301
The enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III have been compared. Both enzymes were found to degrade insulin in such a way that its receptor binding activity was rapidly lost but its precipitability in trichloracetic acid was only slightly decreased. Both enzymes were also found to be inhibited by chelating agents. The bacterial enzyme, which could be purified in large amounts, was found to contain 0.6 mol of zinc per mol of enzyme but no detectable manganese. The mammalian enzyme but not the bacterial one was inhibited by a sulfhydryl alkylating agent. The two enzymes also differed in substrate specificity. The mammalian enzyme degraded insulin much better than insulin-like growth factor II, whereas the bacterial enzyme degraded them equally. The mammalian enzyme could be labeled by cross-linking to insulin = bombyxin II much greater than insulin-like growth factor I and II much greater than relaxin, while the bacterial enzyme was labeled by insulin-like growth factor II greater than insulin = insulin-like growth factor I much greater than relaxin much greater than bombyxin. Finally, sucrose gradient centrifugation and cross-linking studies both in vitro and in vivo indicated that active human enzyme partially existed as a homo- or heterodimer, whereas the bacterial enzyme was active as a monomer.
View details for Web of Science ID A1992HB53200048
View details for PubMedID 1733942
In the last few years several potential substrates of the insulin receptor tyrosine kinase have been identified, purified, and their cDNAs isolated. These putative substrates include: 1) pp15, a fatty acid-binding protein; 2) pp120, a plasma membrane ecto-ATPase; 3) pp42, a MAP serine/threonine kinase; 4) pp85, a subunit of the Type 1 phosphatidylinositol kinase; and 5) pp185, a phosphatidylinositol kinase binding protein. Although the tyrosine phosphorylation of several of these substrates correlates with the signalling capabilities of various mutant receptors, the role of these substrates in mediating any one of insulin's many biological responses is still unknown. In addition, recent data indicate that the tyrosine phosphorylation of pp42 may in fact be due to autophosphorylation, thereby removing it from the list of putative substrates of the insulin receptor kinase. Finally, the present review discusses the question of whether signalling occurs as a result of the tyrosine phosphorylation of substrates or via the formation of signalling complexes.
View details for Web of Science ID A1992HA50400003
View details for PubMedID 1316356
View details for PubMedID 15336083
Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti-insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 monoclonal antibodies that inhibit insulin binding were found to recognize an epitope contained within residues 450-601 of the alpha subunit of the receptor. In contrast, 2 monoclonal antibodies that do not inhibit insulin binding were found to recognize the cysteine-rich region of the alpha subunit. Chimeric insulin receptors that had residues 450-601 replaced with the homologous residues of the insulin-like growth factor I receptor exhibited a decreased ability to bind insulin. In contrast, insulin-like growth factor I receptors that have had the comparable region replaced with that of the insulin receptor showed no decrease in their ability to bind ligand. These results indicate that residues 450-601 of the insulin receptor are important for insulin binding and include the major site for recognition by inhibitory monoclonal antibodies and patients' autoimmune anti-receptor antibodies.
View details for Web of Science ID A1991GM78100101
View details for PubMedID 1719540
Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.
View details for Web of Science ID A1991GB97700009
View details for PubMedID 1651915
We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.
View details for Web of Science ID A1991FN55200001
View details for PubMedID 1645190
Serum-free medium conditioned by BSC-40 cells was analyzed for the presence of transforming growth factor-beta 2 (TGF beta 2)-related proteins. Western blot analysis was performed using site-specific antipeptide antibodies directed against the pro- and mature regions of the TGF beta 2 precursor. When conditioned medium was analyzed by polyacrylamide gel electrophoresis under reducing conditions, proteins with mol wt of 53 kDa (containing both mature and proregion sequences), 34-38 kDa (containing proregion sequences only), and 12 kDa (containing mature sequences) were detected. Under nonreducing conditions, complexes of 60- to 80-kDa, 160- to 200-kDa, as well as 24-kDa mature dimers were seen. Cleavage of mature TGF beta 2 from its precursor was inhibited by monensin and chloroquin, but not by ammonium chloride or methylamine. Two peaks of bioactivity were detected after fractionation on a TSK column corresponding to mol wt of 130 and 400 kDa. These peaks contained TGF beta 2 and pro-TGF beta 2 proteins. Partial purification of the 130-kDa complex followed by N-glyconase digestion indicated that the pro-TGF beta 2 proteins were glycosylated. These data demonstrate that BSC-40 cells secrete mature TGF beta 2 complexed with proregion-containing proteins and suggest that this association may contribute to the latency phenomena observed with respect to this growth regulator.
View details for Web of Science ID A1991FJ39600014
View details for PubMedID 1902166
In the present studies mutant insulin receptors with regulatory tyrosine residues 1162 and 1163 changed to phenylalanines were tested for tyrosine kinase activity. In agreement with prior studies, this mutant receptor was found to exhibit almost no insulin-stimulated exogenous kinase activity when assayed in vitro. In contrast, this mutant receptor was found in situ to have a significant, albeit reduced, ability to mediate the tyrosine phosphorylation of various endogenous proteins, as assessed by Western blotting with antiphosphotyrosine antibodies. In addition, extracts of insulin-treated cells overexpressing this mutant receptor exhibited increased amounts of tyrosine phosphorylated phosphatidylinositol 3-kinase compared to control cells. Finally, this mutant receptor, like the wild-type receptor, was found to mediate an increase in the activity of a membrane-associated phosphatidylinositol 4,5-biphosphate kinase. These results indicate that 1) in vitro assessments of the tyrosine kinase activity of mutant insulin receptors may not accurately reflect their in vivo activities; and 2) the ability of the mutant receptor lacking tyrosine autophosphorylation sites 1162 and 1163 to mediate insulin-stimulated tyrosine phosphorylation of various endogenous substrates may account for the reported ability of this receptor to mediate various biological responses.
View details for Web of Science ID A1991FA90700005
View details for PubMedID 1710030
Autophosphorylation of the insulin receptor has been previously documented to activate the phosphotransferase activity of the receptor from 20- to 200-fold. Biochemical studies have correlated activation of the receptor kinase with the autophosphorylation of tyrosines residues 1158, 1162, and 1163. To further assess the role of these 3 tyrosines in the activation process, we have studied the effect of their substitution with either the neutral amino acids phenylalanine or alanine or with the negatively charged amino acids aspartate and glutamate. In several other proteins, it has been shown that substitution of phosphorylated residues with negatively charged amino acids can mimic the phosphorylation state of the protein. In agreement with previous studies, tyrosines at positions 1162 and 1163 were found to be crucial in the kinase activation process. In contrast, mutant receptors with tyrosine 1158 changed to either phenylalanine or aspartate were still activated to the same extent as the wild-type receptor. An increased basal exogenous kinase activity was observed upon replacement of tyrosines 1162 and 1163 with, in increasing order of potency, aspartate = glutamate less than alanine = phenylalanine. These results indicate that phosphorylation of tyrosines 1162/1163 but not 1158 play a critical role in the activation of the receptor kinase and that the mechanism of activation of the receptor kinase by autophosphorylation is more complex than just an introduction of a cluster of negative charges in this region of the receptor. In addition, the finding of an increased basal kinase activity in receptors lacking tyrosines 1162 and 1163 could explain the reported ability of this receptor to mediate certain biological responses.
View details for Web of Science ID A1991ET17700051
View details for PubMedID 1846000
Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.
View details for Web of Science ID A1991ET74000016
View details for PubMedID 1848562
We have previously shown that the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA binds both IGF-I and II with high affinity. In the present studies, we show that a monoclonal antibody to the IGF-I receptor, alpha IR-3, inhibits the binding of IGF-I but not IGF-II to the expressed receptor in intact cells and after solubilization. Surprisingly, this monoclonal antibody inhibits the ability of both IGF-I and II to stimulate thymidine synthesis in cells with the expressed receptor. Moreover, this antibody inhibits the ability of both IGF-I and II to stimulate the kinase activity of the IGF-I receptor in intact cells. These results indicate that alpha IR-3 binds to the IGF-I receptor in such a way that it does not inhibit the binding of IGF-II but does inhibit the subsequent ability of the receptor to be activated to transmit a signal.
View details for Web of Science ID A1990EA55400050
View details for PubMedID 2171510
Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1990DV41700022
View details for PubMedID 2271531
We have recently described the isolation of a cDNA encoding an enzyme thought to be involved in the degradation of insulin by insulin-responsive tissues. This enzyme, referred to as insulin-degrading enzyme (IDE), is a cytosolic proteinase of 110,000 mol wt which shares structural and functional homology with bacterial protease III. The enzyme may function in the termination of the insulin response. We report here the mapping of the human and mouse IDE genes to human chromosome 10 and mouse chromosome 19, respectively, and evidence for the existence of a single complex IDE gene. We also describe the stable transfection of Chinese hamster ovary cells with a plasmid containing the IDE cDNA under the transcriptional control of the SR alpha promoter. The recombinant protein synthesized by these cells is indistinguishable from the isolated human enzyme in both its size and immunoreactivity and degrades insulin with a specific activity similar to that of the purified proteinase. Overexpression of IDE using this system should allow for a functional test of the role of IDE in insulin action. In addition, expression of various site-directed mutants of IDE will aid in identifying the residues of IDE and protease III that are essential to the activity of this unique family of proteinases.
View details for Web of Science ID A1990DU70300005
View details for PubMedID 2293021
Insulin was found to stimulate the serine/threonine kinase activity of the proto-oncogene product Raf-1. This stimulation was observed in HeLa, NIH 3T3, and Chinese hamster ovary cells, all overexpressing the human insulin receptor. In the HeLa cells, 100 pM insulin gave a significant increase in Raf-1 kinase activity, and 100 nM insulin caused a maximal 2-5-fold increase in activity. The increase in activity was detected after 2 min of insulin treatment and peaked after 5 min. In addition to stimulating Raf-1 kinase activity, insulin caused a shift in the electrophoretic mobility of the Raf-1 protein and an increase in the amount of serine phosphorylation of Raf-1. Moreover, a serine/threonine-specific phosphatase, phosphatase 1, but not two tyrosine-specific phosphatases, was found to deactivate the insulin-activated Raf-1 kinase activity. These findings indicate that insulin activates the serine/threonine kinase activity of the Raf-1 proto-oncogene by increasing its content of phosphoserine.
View details for Web of Science ID A1990DP45800002
View details for PubMedID 2197270
In the present studies, nine different monoclonal antibodies to the extracellular domain of the insulin receptor were tested in three different cell types for their ability to stimulate the intrinsic tyrosine kinase activity of the receptor. Previous studies had suggested that several of these monoclonal antibodies stimulate biological responses without stimulating the intrinsic tyrosine kinase activity of the receptor (Hawley, D. M., Maddux, B. A., Patel, R. G., Wong, K. Y., Manula, P. W., Firestone, G. L., Brunetti, A., Verspohl, E., and Goldfine, I. D. (1989) J. Biol. Chem. 264, 2438-2444 and Soos, M. A., O'Brien, R. M., Brindle, N. P. J., Stigter, J. M., Okamoto, A. K., Whittaker, J., and Siddle, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5217-5221). In the present study, a more sensitive assay was utilized, and these same monoclonal antibodies, when added to intact cells, were found to stimulate the phosphotransferase activity of the receptor. This increase in activity was reversed by phosphatase treatment of the receptor. In contrast, monoclonal antibodies which had no insulin-mimetic activities did not stimulate the receptor's kinase activity. In addition, Western blot analyses of lysates with anti-phosphotyrosine antibodies showed that insulin-mimetic, but not non-insulin-mimetic antibodies, stimulated tyrosine phosphorylation of the receptor as well as an endogenous substrate (phosphoprotein Mr = 160,000). Finally, these antibodies were found to stimulate the tyrosine phosphorylation of another endogenous substrate of the insulin receptor kinase, the type I phosphatidylinositol kinase. These studies support the hypothesis that monoclonal antibodies, like insulin, stimulate biological responses via their ability to stimulate the tyrosine kinase activity of the receptor.
View details for Web of Science ID A1990DF14200077
View details for PubMedID 1693150
Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.
View details for Web of Science ID A1990CM41800008
View details for PubMedID 2153593
The tyrosine kinase activity intrinsic to the insulin receptor is thought to be important in eliciting the intracellular responses to insulin; however, it has been difficult to determine the biochemical functions of the proteins which are substrates for this receptor. Treatment of Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor (CHO.T) with insulin results in a 38 +/- 11 (mean +/- S.E., n = 9)-fold increase in a phosphatidylinositol (PtdIns) kinase activity in anti-phosphotyrosine immunoprecipitates of whole cell lysates. One minute of treatment of cells with insulin causes a dramatic increase in the PtdIns kinase activity in the anti-phosphotyrosine immunoprecipitates; the activity peaks within 5 min and remains elevated for at least 60 min after addition of insulin to the cells. This response is only slightly delayed compared with the time course we observe for activation of the insulin receptor tyrosine kinase. The insulin dose-response curves are also very similar for the activation of the insulin receptor tyrosine kinase activity and for the appearance of PtdIns kinase in the anti-phosphotyrosine immunoprecipitates. Stimulation of the endogenous insulin receptor of CHO cells also results in the association of PtdIns kinase activity with phosphotyrosine-containing proteins. However, CHO cells are less sensitive to insulin than CHO.T cells, and the maximal PtdIns kinase activity in antiphosphotyrosine immunoprecipitates from CHO cells is one-sixth that of CHO.T cells. In contrast, immunoprecipitates from CHO.T cells made with anti-insulin receptor antibodies do not contain significant levels of PtdIns kinase activity. This demonstrates that the PtdIns kinase is either a substrate for the insulin receptor tyrosine kinase or is tightly associated with another tyrosine phosphoprotein, which is not the insulin receptor.
View details for Web of Science ID A1990CF66200060
View details for PubMedID 1688432
The insulin receptor is a large cell surface glycoprotein that concentrates insulin at the site of action and also initiates responses to insulin. The receptor is a disulfide-linked oligomer comprised of two alpha and two beta subunits. Signal transduction through the insulin receptor appears to require the activation of an intrinsic tyrosine-specific protein kinase activity. A variety of disorders, both acquired and genetic, are associated with the development of insulin resistance and are frequently the result of cellular defects in insulin receptor structure, function, and action. The recent cloning of several mutant receptors from patients with genetic forms of extreme insulin resistance has increased our understanding of insulin resistance on the molecular level.
View details for Web of Science ID A1990CX21800011
View details for PubMedID 2184752
View details for Web of Science ID A1990BR15Q00043
Insulin and insulin-like growth factor (IGF)-I inhibit intracellular protein degradation in a variety of different cell types. In the present studies, the IGF-I-induced inhibition of protein metabolism in Chinese hamster ovary (CHO) cells was found to be blocked by polyclonal antibodies to the IGF-II/mannose-6-phosphate phosphate (Man-6-P) receptor, but not by control immunoglobulin. In contrast, these antibodies had no effect on the ability of IGF-I to stimulate glucose uptake in the same cells. The antibodies to the IGF-II/Man-6-P receptor also inhibited the effect of IGF-I and insulin on protein catabolism in human foreskin fibroblasts and human hepatoma cells, respectively. Moreover, CHO cells overexpressing a cDNA coding for the IGF-II/Man-6-P receptor were found to exhibit an increased effect of insulin on protein catabolism. In contrast, the insulin stimulation of glucose uptake is the same in these transfected cells as in the parental CHO cells. These results implicate the IGF-II/Man-6-P receptor in the insulin- and IGF-I-induced inhibition of protein catabolism.
View details for Web of Science ID A1989AB31400003
View details for PubMedID 2544802
Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.
View details for Web of Science ID A1989U182100059
View details for PubMedID 2540751
A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone. A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced. The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein. The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme. The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space. Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling.
View details for Web of Science ID A1988R247900033
View details for PubMedID 3059494
Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.
View details for Web of Science ID A1988Q306000039
View details for PubMedID 2971654
Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.
View details for Web of Science ID A1988P671300076
View details for PubMedID 2969892
The insulin-like growth factor II (IGF-II) is a polypeptide hormone with structural homologies to insulin and insulin-like growth factor I (IGF-I). In contrast to these other hormones, the in vivo function of IGF-II is not known. Although IGF-II can stimulate a broad range of biological responses in isolated cells, these responses have usually been found to be mediated by the insulin and IGF-I receptors. Recently, the receptor for IGF-II was found to also be the receptor for mannose-6-phosphate. Since this latter receptor has been implicated in targeting of lysosomal enzymes, the question is now raised of whether the same protein can also mediate metabolic responses to IGF-II.
View details for Web of Science ID A1988M425400020
View details for PubMedID 2964085
Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.
View details for Web of Science ID A1987L261800042
View details for PubMedID 2962576
The internalization and degradation of insulin was assessed in Chinese hamster ovary cell lines expressing either the wild-type receptor or mutated receptors lacking kinase activity. The mutated receptors included receptors which differed from the wild-type receptor by a single amino acid (substitution of an arginine for lysine at position 1030, a site critical for ATP binding) as well as receptors which had a deletion of 112 amino acids at the carboxyl terminus. Cells expressing mutated receptors lacking kinase activity were found to internalize and degrade insulin at about half the rate of cells expressing wild-type receptors with kinase activity. Moreover, insulin was found incapable of inducing the internalization of the mutated receptors, whereas it could stimulate the internalization of the wild-type receptor. Finally, the constitutive rate of receptor internalization was found to be the same for the mutant and wild-type receptors. These results implicate the intrinsic tyrosine-specific kinase activity of the insulin receptor in the ligand-induced, but not the constitutive, internalization of this receptor.
View details for Web of Science ID A1987K886800006
View details for PubMedID 3316198
Insulin-like growth factor II (IGF-II) shares sequence homology and predicted three-dimensional structure with insulin and IGF-I. IGF-II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF-II. Previous studies have been unable to attribute a particular response of IGF-II through its own receptor. In the present studies, the IGF-II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF-II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF-II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF-I receptors; and (iii) polyclonal antibodies to the IGF-II receptor were found to mimic the ability of IGF-II to stimulate glycogen synthesis. These results indicate that the IGF-II receptor mediates a particular biological response--stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF-II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF-II receptor is not required to stimulate a biological response.
View details for Web of Science ID A1987K569000023
View details for PubMedID 2828025
To test the proposed role of protein disulfide isomerase in the synthesis of immunoglobulins (Ig), intact lymphocytes were treated with a thiol-cleavable, bifunctional cross-linking agent and lysed, and the lysates were immunoprecipitated with antibodies to either Ig or enzyme. When the immunoprecipitates were analyzed on polyacrylamide-sodium dodecyl sulfate gels, protein disulfide isomerase was found to be cross-linked to immunoglobulins. The extent of cross-linking was dependent upon the concentration of cross-linker added and the class of Ig. For IgMs and high concentrations of cross-linker, approximately one molecule of Ig was coupled per two molecules of enzyme. For IgGs, the extent of cross-linking was less. Finally, depletion of the intracellularly reduced glutathione by diamide was found to also result in the linkage of protein disulfide isomerase to IgM. These results therefore support the hypothesis that protein disulfide isomerase functions in the in vivo synthesis of immunoglobulins.
View details for Web of Science ID A1987J320400001
View details for PubMedID 3663584
Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.
View details for Web of Science ID A1986C835000009
View details for PubMedID 2424018
The insulin receptor tyrosine kinase is required for insulin to elicit subsequent biological signalling. Recent studies have identified several endogenous substrates of the insulin receptor kinase, including one called insulin receptor substrate 1 (IRS-1). Tyrosine phosphorylation of this substrate results in its being bound by various proteins containing src homology 2 (SH2) sites, including a phosphatidylinositol 3-kinase and a ras activator complex containing GRB2 and son of sevenless (SOS) 1. Decreases in the insulin receptor tyrosine kinase activity have been observed in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. A model of insulin resistance has recently been described in which the insulin receptor is expressed in Chinese hamster ovary cells along with the phospholipid- and calcium-activated serine/threonine kinase called protein kinase C. In this model system, activation of protein kinase C is shown to interfere with insulin receptor signalling by inhibiting tyrosine phosphorylation of IRS-1 and its subsequent binding by phosphatidylinositol 3-kinase. Such a model system may be further utilized to determine the detailed biochemical basis for insulin resistance.
View details for Web of Science ID A1994NV61500014
View details for PubMedID 8088704
View details for Web of Science ID A1990BQ80E00007