Hyun-A Seong

Critical Role of Cysteine Residue 81 of Macrophage Migration Inhibitory Factor (MIF) in MIF-induced Inhibition of p53 Activity

Macrophage migration inhibitory factor (MIF) is a potent modulator of the p53 signaling pathway, but the molecular mechanisms of the effect of MIF on p53 function have so far remained unclear. Here we show that MIF physically interacts with the p53 tumor suppressor in vitro and in vivo. This association was significantly reduced by a C81S mutation but not C57S or C60S mutations, suggesting that Cys81 is essential for the in vivo association between MIF and p53. This association also depended on Cys242 (and, to some extent, on Cys238) within the central DNA binding domain of p53. Ectopic expression of MIF, but not MIF(C81S), inhibited p53-mediated transcriptional activation in a dose-dependent manner. Conversely, knockdown of endogenous MIF stimulated p53-mediated transcription. MIF inhibited p53-induced apoptosis and cell cycle arrest, whereas the MIF(C81S) mutant, which is unable to physically associate with p53, had no effect. Consistent with these findings, confocal microscopy showed that MIF prevented p53 translocation from the cytoplasm to the nucleus. We also demonstrated that MIF suppresses p53 activity by stabilizing the physical association between p53 and Mdm2. These results suggest that MIF physically associates with p53 and negatively regulates p53 function.

NM23-H1 Tumor Suppressor and Its Interacting Partner STRAP Activate p53 Function *

p53 plays a critical role in a variety of growth inhibitory responses, including cell cycle arrest, differentiation, and apoptosis, and contributes to tumor suppression. Here we show that NM23-H1 and its binding partner STRAP (serine-threonine kinase receptor-associated protein) interact with p53 and potentiate p53 activity. Both NM23-H1 and STRAP directly interact with the central DNA binding domain within residues 113-290. The use of NM23-H1 and STRAP mutants revealed that Cys145 of NM23-H1 and Cys152 (or Cys270) of STRAP were responsible for p53 binding. Furthermore, Cys176 and Cys135 of p53 were required to bind NM23-H1 and STRAP, respectively. Ectopic expression of wild-type NM23-H1 and STRAP, but not NM23-H1(C145S) and STRAP(C152S/C270S), positively regulated p53-mediated transcription in a dose-dependent manner. Knockdown of endogenous NM23-H1 or STRAP produced an opposite trend and inhibited the p53-mediated transcription. Similarly, NM23-H1 and STRAP stimulated p53-induced apoptosis and growth inhibition, whereas the NM23-H1(C145S) and STRAP(C152S/C270S) mutants had no effect. We also demonstrated that p53 activation by NM23-H1 and STRAP was mediated by removing Mdm2, a negative regulator of p53, from the p53-Mdm2 complex. These results suggest that NM23-H1 and its interacting partner STRAP physically interact with p53 and positively regulate its functions, including p53-induced apoptosis and cell cycle arrest.

NM23-H1 Tumor Suppressor Physically Interacts with Serine-Threonine Kinase Receptor-associated Protein, a Transforming Growth Factor-β (TGF-β) Receptor-interacting Protein, and Negatively Regulates TGF-β Signaling

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-β receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys145 of NM23-H1 and Cys152 and Cys270 of STRAP but did not appear to involve Cys4 and Cys109 of NM23-H1, suggesting that a disulfide linkage involving Cys145 of NM23-H1 and Cys152 or Cys270 of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or β-mercaptoethanol but not H2O2. Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-β signaling in a dose-dependent manner, enhanced stable association between the TGF-β receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-β-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-β signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-β-dependent biological activity as well as NM23-H1 activity.

Regulation of Transforming Growth Factor-β Signaling and PDK1 Kinase Activity by Physical Interaction between PDK1 and Serine-Threonine Kinase Receptor-associated Protein

To gain more insights about the biological roles of PDK1, we have used the yeast two-hybrid system and in vivo binding assay to identify interacting molecules that associate with PDK1. As a result, serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor-β (TGF-β) receptor-interacting protein, was identified as an interacting partner of PDK1. STRAP was found to form in vivo complexes with PDK1 in intact cells. Mapping analysis revealed that this binding was only mediated by the catalytic domain of PDK1 and not by the pleckstrin homology domain. Insulin enhanced a physical association between PDK1 and STRAP in intact cells, but this insulin-induced association was prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. In addition, the association between PDK1 and STRAP was decreased by TGF-β treatment. Analysis of the activities of the interacting proteins showed that PDK1 kinase activity was significantly increased by coexpression of STRAP, probably through the inhibition of the binding of 14-3-3, a negative regulator, to PDK1. Consistently, knockdown of the endogenous STRAP by the transfection of the small interfering RNA resulted in the decrease of PDK1 kinase activity. PDK1 also exhibited an inhibition of TGF-β signaling with STRAP by contributing to the stable association between TGF-β receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-β. Knockdown of endogenous PDK1 with small interfering RNA has an opposite effect. Taken together, these results suggested that STRAP acts as an intermediate signaling molecule linking between the phosphatidylinositol 3-kinase/PDK1 and the TGF-β signaling pathways.

Murine Protein Serine/Threonine Kinase 38 Activates Apoptosis Signal-regulating Kinase 1 via Thr838 Phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H2O2 or tumor necrosis factor α treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr838 phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H2O2-mediated apoptosis by enhancing the ASK1 activity through Thr838 phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.

Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38).

We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295-301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7 kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen,liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.

SMILE, a new orphan nuclear receptor SHP-interacting protein, regulates SHP-repressed estrogen receptor transactivation

SHP (small heterodimer partner) is a well-known NR (nuclear receptor) co-regulator. In the present study, we have identified a new SHP-interacting protein, termed SMILE (SHP-interacting leucine zipper protein), which was previously designated as ZF (Zhangfei) via a yeast two-hybrid system. We have determined that the SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE)]. Mutational analysis has demonstrated that the SMILE isoforms arise from the alternative usage of initiation codons. We have confirmed the in vivo interaction and co-localization of the SMILE isoforms and SHP. Domain-mapping analysis indicates that the entire N-terminus of SHP and the middle region of SMILE-L are involved in this interaction. Interestingly, the SMILE isoforms counteract the SHP repressive effect on the transactivation of ERs (estrogen receptors) in HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40), but enhance the SHP-repressive effect in MCF-7, T47D and MDA-MB-435 cells. Knockdown of SMILE gene expression using siRNA (small interfering RNA) in MCF-7 cells increases ER-mediated transcriptional activity. Moreover, adenovirus-mediated overexpression of SMILE and SHP down-regulates estrogen-induced mRNA expression of the critical cell-cycle regulator E2F1. Collectively, these results indicate that SMILE isoforms regulate the inhibition of ER transactivation by SHP in a cell-type-specific manner and act as a novel transcriptional co-regulator in ER signalling.

Direct Interaction between NM23-H1 and Macrophage Migration Inhibitory Factor (MIF) Is Critical for Alleviation of MIF-mediated Suppression of p53 Activity

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that is involved in host immune and inflammatory responses, as well as tumorigenesis. However, the regulatory mechanism of MIF function is unclear. Here we report that the NM23-H1 interacts with MIF in cells, as demonstrated by cotransfection and coimmunoprecipitation experiments. Analysis of cysteine (Cys) to serine (Ser) substitution mutants of NM23-H1 (C4S, C109S, and C145S) and MIF (C57S, C60S, and C81S) revealed that Cys145 of NM23-H1 and Cys60 of MIF are responsible for complex formation. NM23-H1-MIF complexes were dependent on reducing conditions, such as the presence of dithiothreitol or β-mercaptoethanol, but not H2O2. NM23-H1 alleviated the MIF-mediated suppression of p53-induced apoptosis and cell cycle arrest by promoting the dissociation of MIF from MIF-p53 complexes. In addition, NM23-H1 significantly inhibited the MIF-induced proliferation of quiescent NIH 3T3 cells through a direct interaction with MIF, and decreased the MIF-induced activation of phosphatidylinositol 3-kinase/PDK1 and p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinase. The results of the current study suggest that the NM23-H1 functions as a negative regulator of MIF.

3-Phosphoinositide-dependent PDK1 Negatively Regulates Transforming Growth Factor-β-induced Signaling in a Kinase-dependent Manner through Physical Interaction with Smad Proteins

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-β (TGF-β) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-β signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-β treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-β-induced transcription, as well as TGF-β-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-β. Taken together, our results suggest that PDK1 negatively regulates TGF-β-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Murine protein serine/threonine kinase 38 stimulates TGF-β signaling in a kinase-dependent manner via direct phosphorylation of Smad proteins

The present study demonstrated that murine protein serine/threonine kinase 38 (MPK38) coimmunoprecipitates with Smad proteins (Smad2, -3, -4, and -7) and that this association is mediated by the catalytic kinase domain of MPK38. The association between MPK38 and Smad2, -3, and -4 was significantly increased by TGF-β or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-β-induced transcription required for TGF-β-mediated biological functions, such as apoptosis and cell growth arrest, in a kinase-dependent manner. Knockdown of endogenous MPK38 showed an opposite effect, inhibiting TGF-β signaling. MPK38-mediated phosphorylation of Smad proteins (Ser245 of Smad2, Ser204 of Smad3, Ser343 of Smad4, and Thr96 of Smad7) was also found to be crucial to the positive regulation of TGF-β signaling induced by MPK38. In addition, MPK38 enhanced nuclear translocation of Smad3, as well as redistribution of Smad7 from the nucleus to the cytoplasm, in response to TGF-β. Together, these results indicate that MPK38 functions as a stimulator of TGF-β signaling through direct interaction with and phosphorylation of Smad proteins.