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 H 2 O 2 or tumor necrosis factor ␣ 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 Thr 838 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 H 2 O 2 -mediated apoptosis by enhancing the ASK1 activity through Thr 838 phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) 2 is one of the mitogen-activated protein kinase kinase kinases (MAPKKK) that is stimulated in response to various cellular stresses, including reactive oxygen species, tumor necrosis factor ␣ (TNF-␣), Fas, ischemia insult, and anti-tumor agents. ASK1 stimulation leads to activation of the c-Jun NH 2 -terminal kinase (JNK)/p38 signaling cascade by phosphorylating and activating mitogen-activated protein kinase kinases (MAPKK) such as MKK3, -4, -6, and -7 (1-3). Emerging evidence indicates that ASK1 activity is regulated by its interaction with several cellular partners (3-8), including thioredoxin (Trx), glutaredoxin, heat shock protein 72 (Hsp72), 14-3-3, and protein serine/threonine phosphatase 5 (PP5). For example, Trx and glutaredoxin bind to the NH 2 -and COOH-terminal domains of ASK1, respectively, and inhibit ASK1 kinase activity, and Hsp72 inhibits ASK1 activation through direct interaction. These findings suggest that other ASK1-interacting proteins could be involved in the regulation of ASK1 activity.Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase-related serine/ threonine kinase family (9, 10). MPK38 was originally identified as a murine counterpart for its human homolog, HPK38/ hMelk/KIAA175, that may be involved in the proliferation of interleukin-4-induced normal human keratinocytes (9). The importance of MPK38 in oncogenesis is also underscored by the finding that MPK38 expression is increased in tumor-derived progenitor cells as well as in cancers of nondifferentiated cells (11-13). However, the physiological regulation and functions of MPK38 have r...

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.

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor and acts as a coregulator of various nuclear receptors. Herein, we examined a novel cross talk between SHP and a forkhead transcription factor HNF3 (hepatocyte nuclear factor 3/Foxa. Transient transfection assay demonstrated that SHP inhibited the transcriptional activity of all three isoforms of HNF3, HNF3alpha, beta, and gamma. In vivo and in vitro protein interaction studies showed that SHP physically interacted with HNF3. Adenovirus-mediated overexpression of SHP significantly decreased the mRNA levels of glucose-6-phosphase (G6Pase), cholesterol 7-alpha-hydroxylase (CYP7A1), and phosphoenolpyruvate carboxykinase (PEPCK) in HepG2 cells and rat primary hepatocytes. Moreover, the mRNA level of G6Pase was notably increased by down-regulation of SHP with small interfering RNA. Interestingly, HNF3 transactivity was still repressed by SHPDelta128-139 that fails to repress nuclear receptors. Mapping of interaction domain revealed that SHP interacted with forkhead DNA binding domain of HNF3alpha. Gel mobility shift and chromatin immunoprecipitation assays demonstrated that SHP inhibits DNA binding of HNF3. These results suggest that SHP is involved in the regulation of G6Pase, CYP7A1, and PEPCK gene expression via novel mechanism of inhibition of HNF3 activity and expand the role of SHP as a coregulator of other family of transcription factors in addition to nuclear receptors.

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-␤ (TGF-␤) 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-␤ 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-␤ signaling with STRAP by contributing to the stable association between TGF-␤ receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-␤. 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-␤ signaling pathways.

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) . Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-␤ signaling in a dose-dependent manner, enhanced stable association between the TGF-␤ receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-␤-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-␤ 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-␤-dependent biological activity as well as NM23-H1 activity.-The NM23 family of genes is characterized by reduced expression in certain highly metastatic cell lines and tumors (1).In humans, the eight NM23 genes that have been identified to date, NM23-H1, , and NM23-H8, encode NDP kinases or homologous isoforms (2). However, although NM23-H1 was initially identified as a putative metastasis suppressor, its enzymatic activity does not appear to be responsible for its function as a metastasis suppressor during tumor progression (3). Studies of NM23 family proteins in other species have provided evidence for their role in proliferation, differentiation, apoptosis, development, and endocytosis (4). In Drosophila, for example, abnormal wing discs (awd) is an NDP kinase, and the killerof-prune mutation of awd (awd k-pn ) causes abnormalities in cell morphology and differentiation (5). Recently, both NM23-H1 and NM23-H2 have been reported to play a role in endocytosis (6). In addition, NM23 has been associated with the differentiation of human MDA-MB-435 breast carcinoma cells (7). The ability of NM23 family proteins to regulate such a diverse set of cellular processes has recently been linked to their ability to modulate signal transduction by a diverse set of growth factors, such as transforming growth factor-␤1 (TGF-␤1), 2 nerve growth factor, platelet-derived growth factor, and insulin-like growth factor-1 (8). However, the mechanism of regulation of these signaling pathways by NM23 family proteins is unknown. To date, NM23 family proteins have been shown to associate with several cellular proteins, including glyceraldehyde-3-phosphate dehydrogenase (9), Hsc70 (70-kDa heat shock cognate protein) (10), telomere (11), ROR␣ (retinoid acid receptor-related orphan receptor ␣)/RZR␤ (retinoid Z receptor ␤) (12), Rad, a Ras-related small GTPase (13), creatine kinase and antioxidant protein (14), and thromboxane A2 receptor, a G protein-coupled receptor (6). These results suggest that the identification of additional binding partners of NM23 proteins will provide gre...