Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.
View details for DOI 10.1074/jbc.M400962200
View details for Web of Science ID 000220870400090
View details for PubMedID 14761958
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
View details for PubMedID 12840518
PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.
View details for Web of Science ID A1992HV22300006
View details for PubMedID 1318025
A monoclonal antibody has been developed against the type II PtdIns 4-kinase from bovine brain. This antibody, 4C5G, causes greater than 90% inhibition of the type II PtdIns 4-kinase from bovine brain, rat brain and human erythrocytes. However, it fails to inhibit type III PtdIns 4-kinase from bovine brain or PtdIns 3-kinase from rat liver. These results suggest that type II and type III PtdIns 4-kinases are distinct gene products, and that 4C5G will be useful in studying the function of the type II PtdIns 4-kinase.
View details for Web of Science ID A1991EU32800006
View details for PubMedID 1846531
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