Bio

Professional Education


  • PhD, Pohang University of Science and Technology, Signaling Proteome (2006)

Stanford Advisors


Publications

Journal Articles


  • Leucyl-tRNA Synthetase Is an Intracellular Leucine Sensor for the mTORC1-Signaling Pathway CELL Han, J. M., Jeong, S. J., Park, M. C., Kim, G., Kwon, N. H., Kim, H. K., Ha, S. H., Ryu, S. H., Kim, S. 2012; 149 (2): 410-424

    Abstract

    Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase, which regulates protein translation, cell size, and autophagy. However, the amino acid sensor that directly couples intracellular amino acid-mediated signaling to mTORC1 is unknown. Here we show that leucyl-tRNA synthetase (LRS) plays a critical role in amino acid-induced mTORC1 activation by sensing intracellular leucine concentration and initiating molecular events leading to mTORC1 activation. Mutation of LRS amino acid residues important for leucine binding renders the mTORC1 pathway insensitive to intracellular levels of amino acids. We show that LRS directly binds to Rag GTPase, the mediator of amino acid signaling to mTORC1, in an amino acid-dependent manner and functions as a GTPase-activating protein (GAP) for Rag GTPase to activate mTORC1. This work demonstrates that LRS is a key mediator for amino acid signaling to mTORC1.

    View details for DOI 10.1016/j.cell.2012.02.044

    View details for Web of Science ID 000302805600019

    View details for PubMedID 22424946

  • Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host-guest binding pair NATURE CHEMISTRY Lee, D., Park, K. M., Banerjee, M., Ha, S. H., Lee, T., Suh, K., Paul, S., Jung, H., Kim, J., Selvapalam, N., Ryu, S. H., Kim, K. 2011; 3 (2): 154-159

    Abstract

    Membrane proteomics, the large-scale global analysis of membrane proteins, is often constrained by the efficiency of separating and extracting membrane proteins. Recent approaches involve conjugating membrane proteins with the small molecule biotin and using the receptor streptavidin to extract the labelled proteins. Despite the many advantages of this method, several shortcomings remain, including potential contamination by endogenously biotinylated molecules and interference by streptavidin during analytical stages. Here, we report a supramolecular fishing method for membrane proteins using the synthetic receptor-ligand pair cucurbit[7]uril-1-trimethylammoniomethylferrocene (CB[7]-AFc). CB[7]-conjugated beads selectively capture AFc-labelled proteins from heterogeneous protein mixtures, and AFc-labelling of cells results in the efficient capture of membrane proteins by these beads. The captured proteins can be recovered easily at room temperature by treatment with a strong competitor such as 1,1'-bis(trimethylammoniomethyl)ferrocene. This synthetic but biocompatible host-guest system may be a useful alternative to streptavidin-biotin for membrane proteomics as well as other biological and biotechnological applications.

    View details for DOI 10.1038/NCHEM.928

    View details for Web of Science ID 000286505700013

    View details for PubMedID 21258389

  • Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D MOLECULAR AND CELLULAR BIOLOGY Kim, H. W., Ha, S. H., Lee, M. N., Huston, E., Kim, D., Jang, S. K., Suh, P., Houslay, M. D., Ryu, S. H. 2010; 30 (22): 5406-5420

    Abstract

    The mammalian target of rapamycin complex 1 (mTORC1) is a molecular hub that regulates protein synthesis in response to a number of extracellular stimuli. Cyclic AMP (cAMP) is considered to be an important second messenger that controls mTOR; however, the signaling components of this pathway have not yet been elucidated. Here, we identify cAMP phosphodiesterase 4D (PDE4D) as a binding partner of Rheb that acts as a cAMP-specific negative regulator of mTORC1. Under basal conditions, PDE4D binds Rheb in a noncatalytic manner that does not require its cAMP-hydrolyzing activity and thereby inhibits the ability of Rheb to activate mTORC1. However, elevated cAMP levels disrupt the interaction of PDE4D with Rheb and increase the interaction between Rheb and mTOR. This enhanced Rheb-mTOR interaction induces the activation of mTORC1 and cap-dependent translation, a cellular function of mTORC1. Taken together, our results suggest a novel regulatory mechanism for mTORC1 in which the cAMP-determined dynamic interaction between Rheb and PDE4D provides a key, unique regulatory event. We also propose a new role for PDE4 as a molecular transducer for cAMP signaling.

    View details for DOI 10.1128/MCB.00217-10

    View details for Web of Science ID 000283656700013

    View details for PubMedID 20837708

  • Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis PROTEOMICS Kim, J., Choi, Y. S., Lim, S., Yea, K., Yoon, J. H., Jun, D., Ha, S. H., Kim, J., Kim, J. H., Suh, P., Ryu, S. H., Lee, T. G. 2010; 10 (3): 394-405

    Abstract

    Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label-free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT-PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor-beta (TGF-beta) signaling changed during adipogenesis. The expressions of secreted frizzled-related proteins, dickkopf-related proteins, and latent TGF-beta-binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF-beta signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity-related metabolic diseases.

    View details for DOI 10.1002/pmic.200900218

    View details for Web of Science ID 000274707400004

    View details for PubMedID 19953544

  • Collapsin response mediator protein-2 regulates neurite formation by modulating tubulin GTPase activity CELLULAR SIGNALLING Chae, Y. C., Lee, S., Heo, K., Ha, S. H., Jung, Y., Kim, J. H., Ihara, Y., Suh, P., Ryu, S. H. 2009; 21 (12): 1818-1826

    Abstract

    Collapsin response mediator protein-2 (CRMP-2) plays a key role in axonal development by regulating microtubule dynamics. However, the molecular mechanisms underlying this function have not been clearly elucidated. In this study, we demonstrated that hCRMP-2, specifically amino acid residues 480-509, is essential for stimulating tubulin GTPase activity. We also found that the GTPase-activating protein (GAP) activity of hCRMP-2 was important for microtubule assembly and neurite formation in differentiated PC12 pheochromocytoma cell lines. Mutant hCRMP-2, lacking arginine residues responsible for GAP activity, inhibited microtubule assembly and neurite formation. Interestingly, we found that the N-terminal region (amino acids150-299) of hCRMP-2 had an inhibitory role on GAP activity via a direct interaction with the C-terminal region (amino acids 480-509). Our results suggest that CRMP-2 as a tubulin direct binder may be a GAP of tubulin in neurite formation and that its GAP activity may be regulated by an intramolecular interaction with an N-terminal inhibitory region.

    View details for DOI 10.1016/j.cellsig.2009.07.017

    View details for Web of Science ID 000271269900011

    View details for PubMedID 19666111

  • Phosphorylation of Phospholipase C-delta(1) Regulates its Enzymatic Activity JOURNAL OF CELLULAR BIOCHEMISTRY Fujii, M., Yi, K. S., Kim, M. J., Ha, S. H., Ryu, S. H., Suh, P., Yagisawa, H. 2009; 108 (3): 638-650

    Abstract

    Phosphorylation of phospholipase C-delta(1) (PLC-delta(1)) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC-delta(1) most potently. It was also demonstrated that PLC-delta(1) directly bound PKC-alpha via its pleckstrin homology (PH) domain. Using deletion mutants of PLC-delta(1) and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC-delta(1). In vitro phosphorylation of PLC-delta(1) by PKC stimulated [(3)H]PtdIns(4,5)P(2) hydrolyzing activity and [(3)H]Ins(1,4,5)P(3)-binding of the PLC-delta(1). On the other hand, endogenous PLC-delta(1) was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we determined that Thr209 of PLC-delta(1) is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC-delta(1) was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC-alpha reduced serine phosphorylation of PLC-delta(1) detected by an anti-phosphoserine antibody and PLC-delta(1)-dependent basal production of inositol phosphates in NIH-3T3 cells, suggesting PKC-alpha activates phosphatase or inactivates another kinase involved in PLC-delta(1) serine phosphorylation to modulate the PLC-delta(1) activity in vivo. Taken together, these results suggest that PLC-delta(1) has multiple phosphorylation sites and phosphorylation status of PLC-delta(1) regulates its activity positively or negatively depends on the phosphorylation sites.

    View details for DOI 10.1002/jcb.22297

    View details for Web of Science ID 000270567100011

    View details for PubMedID 19681039

  • Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb MOLECULAR AND CELLULAR BIOLOGY Lee, M. N., Ha, S. H., Kim, J., Koh, A., Lee, C. S., Kim, J. H., Jeon, H., Kim, D., Suh, P., Ryu, S. H. 2009; 29 (14): 3991-4001

    Abstract

    The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR's access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.

    View details for DOI 10.1128/MCB.00165-09

    View details for Web of Science ID 000267939300015

    View details for PubMedID 19451232

  • PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals CELLULAR SIGNALLING Ha, S. H., Kim, D., Kim, I., Kim, J. H., Lee, M. N., Lee, H. J., Kim, J. H., Jang, S. K., Suh, P., Ryu, S. H. 2006; 18 (12): 2283-2291

    Abstract

    Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265-269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.

    View details for DOI 10.1016/j.cellsig.2006.05.021

    View details for Web of Science ID 000242266400022

    View details for PubMedID 16837165

  • RGS2 promotes formation of neurites by stimulating microtubule polymerization CELLULAR SIGNALLING Heo, K., Ha, S. H., Chae, Y. C., Lee, S., Oh, Y., Kim, Y., Kim, S., Kim, J. H., Mizoguchi, A., Itoh, T. J., Kwon, H. M., Ryu, S. H., Suh, P. 2006; 18 (12): 2182-2192

    Abstract

    Regulator of G-protein signaling (RGS) proteins interact with alpha subunits of heterotrimeric G-proteins via the RGS domain and attenuate their activity by accelerating GTPase activity. RGS2, a member of the RGS family, regulates synaptic development via hereto unknown mechanism. In this study, we found that RGS2 directly interacted with tubulin via a short region at the N-terminus: amino acids 41-60. RGS2 enhanced microtubule polymerization in vitro, and the tubulin binding region was necessary and sufficient for this activity. In Vero cells, polymerization of microtubule was stimulated when peptides containing the tubulin binding region were microinjected. Immunocytochemical analysis showed that endogenous RGS2 was localized at the termini of neurites in differentiated PC12 cells. Over-expression of RGS2 enhanced the nerve growth factor-induced neurite outgrowth in PC12 cells, while specific knock-down of endogenous RGS2 suppressed the neurite outgrowth. These findings demonstrate that RGS2 contributes to the neuronal cell differentiation via regulation of microtubule dynamics.

    View details for DOI 10.1016/j.cellsig.2006.05.006

    View details for Web of Science ID 000242266400012

    View details for PubMedID 16820281

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