Sang Hoon Ha

Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

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 mTORs access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.

A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon.

ABSTRACT Translational initiation of hepatitis C virus (HCV) mRNA occurs by internal entry of ribosomes into an internal ribosomal entry site (IRES) at the 5′ nontranslated region. A region encoding the N-terminal part of the HCV polyprotein has been shown to augment the translation of HCV mRNA. Here we show that a cellular protein, NS1-associated protein 1 (NSAP1), augments HCV mRNA translation through a specific interaction with an adenosine-rich protein-coding region within the HCV mRNA. The overexpression of NSAP1 specifically enhanced HCV IRES-dependent translation, and knockdown of NSAP1 by use of a small interfering RNA specifically inhibited the translation of HCV mRNA. An HCV replicon RNA capable of mimicking the HCV proliferation process in host cells was further used to confirm that NSAP1 enhances the translation of HCV mRNA. These results suggest the existence of a novel mechanism of translational enhancement that acts through the interaction of an RNA-binding protein with a protein coding sequence.

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis.

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‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β 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.

Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D

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.

Hydrogen peroxide induces association between glyceraldehyde 3-phosphate dehydrogenase and phospholipase D2 to facilitate phospholipase D2 activation in PC12 cells

Oxidative stress or signaling is widely implicated in apoptosis, ischemia and mitogenesis. Previously, our group reported that the hydrogen peroxide (H2O2)‐dependent activation of phospholipase D2 (PLD2) in PC12 cells is involved in anti‐apoptotic effect. However, the precise mechanism of PLD2 activation by H2O2 was not revealed. To find H2O2‐dependent PLD2‐regulating proteins, we immunoprecipitated PLD2 from PC12 cells and found that glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) coimmunoprecipitated with PLD2 upon H2O2 treatment. This interaction was found to be direct by in vitro reconstitution of purified GAPDH and PLD2. In vitro studies also indicated that PLD2‐associated GAPDH was modified on its reactive cysteine residues. Koningic acid, an alkylator of GAPDH on catalytic cysteine residue, also increased interaction between the two proteins in vitro and enhanced PLD2 activity in PC12 cells. Blocking H2O2‐dependent modification of GAPDH with 3‐aminobenzamide resulted in the inhibition of the GAPDH/PLD2 interaction and attenuated H2O2‐induced PLD2 activation in PC12 cells. From the results, we suggest that H2O2 modifies GAPDH on its catalytic cysteine residue not only to inactivate the dehydrogenase activity of GAPDH but also to endow GAPDH with the ability to bind PLD2 and the resulting association is involved in the regulation of PLD2 activity by H2O2.

Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

ABSTRACT Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.

Phosphorylation of Phospholipase C‐δ1 Regulates its Enzymatic Activity

Phosphorylation of phospholipase C‐δ1 (PLC‐δ1) 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‐δ1 most potently. It was also demonstrated that PLC‐δ1 directly bound PKC‐α via its pleckstrin homology (PH) domain. Using deletion mutants of PLC‐δ1 and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC‐δ1. In vitro phosphorylation of PLC‐δ1 by PKC stimulated [3H]PtdIns(4,5)P2 hydrolyzing activity and [3H]Ins(1,4,5)P3‐binding of the PLC‐δ1. On the other hand, endogenous PLC‐δ1 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‐δ1 is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC‐δ1 was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC‐α reduced serine phosphorylation of PLC‐δ1 detected by an anti‐phosphoserine antibody and PLC‐δ1‐dependent basal production of inositol phosphates in NIH‐3T3 cells, suggesting PKC‐α activates phosphatase or inactivates another kinase involved in PLC‐δ1 serine phosphorylation to modulate the PLC‐δ1 activity in vivo. Taken together, these results suggest that PLC‐δ1 has multiple phosphorylation sites and phosphorylation status of PLC‐δ1 regulates its activity positively or negatively depends on the phosphorylation sites. J. Cell. Biochem. 108: 638–650, 2009.

Nudix-type motif 2 contributes to cancer proliferation through the regulation of Rag GTPase-mediated mammalian target of rapamycin complex 1 localization

Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control.

Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.