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. 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. IntroductionType I transforming growth factor-β (TGF-β) receptor kinases are serine/threonine kinases with roles in a wide array of cellular processes. Phosphorylation of the C-terminal SXS motif in receptor-regulated Smads (R-Smads) by these kinases is a crucial step in TGF-β family signaling (1Abdollah S. Macías-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 2Macías-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 3Souchelnytskyi S. Tamaki K. Engström U. Wernstedt C. ten Dijke P. Heldin C.H. J. Biol. Chem. 1997; 272: 28107-28115Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). R-Smad phosphorylation results in the formation of heterodimeric complexes with the common Smad, Smad4. In addition, the phosphorylation of serine or threonine residues in the N-terminal or linker region has been observed in endogenous R-Smads (4Xu L. Biochim. Biophys. Acta. 2006; 1759: 503-513Crossref PubMed Scopus (77) Google Scholar). Several lines of evidence have demonstrated the existence of kinases responsible for N-terminal or linker region phosphorylation events, including mitogen-activated protein kinases (MAPKs), Ca2+ and calmodulin-dependent kinase II, cyclin-dependent kinase (CDK), 3The abbreviations used are: CDKcyclin-dependent kinaseSmadSma- and Mad-related proteinI-Smadinhibitory SmadMHMad homologyTβRtransforming growth factor-β receptorZPR9zinc-finger-like protein 9MPK38murine protein serine/threonine kinase 38PDK13-phosphoinositide-dependent protein kinase 1. protein kinase C (PKC), and G protein-coupled receptor kinase 2 (5Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar). Common Smad, Smad4, is constitutively phosphorylated in cells, although not all of the phosphorylation sites are known. Extracellular signal-regulated kinase (ERK) has been shown to phosphorylate Smad4 at Thr277, resulting in the prevention of nuclear translocation of Smad4 (6Roelen B.A. Cohen O.S. Raychowdhury M.K. Chadee D.N. Zhang Y. Kyriakis J.M. Alessandrini A.A. Lin H.Y. Am. J. Physiol. Cell Physiol. 2003; 285: C823-C830Crossref PubMed Scopus (50) Google Scholar). The inhibitory Smads (I-Smads), Smad6 and Smad7, are phosphorylated by uncharacterized kinases (7Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 8Pulaski L. Landström M. Heldin C.H. Souchelnytskyi S. J. Biol. Chem. 2001; 276: 14344-14349Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This provides a mechanism for integration of the Smad pathway with other signaling pathways, in which kinases act as essential mediators, which modulate Smad-mediated signaling. Thus, the identification and characterization of additional cellular kinases responsible for Smad phosphorylation could lead to a better understanding of the regulatory role of Smad phosphorylations, especially in the N-terminal and linker region, in TGF-β signaling.Murine protein serine/threonine kinase 38 (MPK38), also known as pEg3 kinase and maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase family of serine-threonine kinases (9Gil M. Yang Y. Lee Y. Choi I. Ha H. Gene. 1997; 195: 295-301Crossref PubMed Scopus (51) Google Scholar, 10Heyer B.S. Warsowe J. Solter D. Knowles B.B. Ackerman S.L. Mol. Reprod. Dev. 1997; 47: 148-156Crossref PubMed Google Scholar). The expression patterns of MPK38 during maturation of oocytes and pre-implantation development were found to be typical of that described for maternally expressed genes, including β-actin and E-cadherin (11Bachvarova R. Cohen E.M. De Leon V. Tokunaga K. Sakiyama S. Paynton B.V. Development. 1989; 106: 561-565Crossref PubMed Google Scholar, 12Ohsugi M. Hwang S.Y. Butz S. Knowles B.B. Solter D. Kemler R. Dev. Dyn. 1996; 206: 391-402Crossref PubMed Scopus (78) Google Scholar). This suggested that MPK38 may play a role in transducing signals in embryonic cells during pre-implantation, although its exact roles in development were not revealed. Additionally, MPK38 is also expressed in several other tissues, including embryonic stem cells (13Nakano I. Paucar A.A. Bajpai R. Dougherty J.D. Zewail A. Kelly T.K. Kim K.J. Ou J. Groszer M. Imura T. Freije W.A. Nelson S.F. Sofroniew M.V. Wu H. Liu X. Terskikh A.V. Geschwind D.H. Kornblum H.I. J. Cell Biol. 2005; 170: 413-427Crossref PubMed Scopus (117) Google Scholar), adult germ cells (14Heyer B.S. Kochanowski H. Solter D. Dev. Dyn. 1999; 215: 344-351Crossref PubMed Scopus (46) Google Scholar), hematopoietic stem cells (15Saito R. Tabata Y. Muto A. Arai K. Watanabe S. Mol. Cell Biol. 2005; 25: 6682-6693Crossref PubMed Scopus (19) Google Scholar), adult hematopoietic cells (9Gil M. Yang Y. Lee Y. Choi I. Ha H. Gene. 1997; 195: 295-301Crossref PubMed Scopus (51) Google Scholar), and neural stem cells (13Nakano I. Paucar A.A. Bajpai R. Dougherty J.D. Zewail A. Kelly T.K. Kim K.J. Ou J. Groszer M. Imura T. Freije W.A. Nelson S.F. Sofroniew M.V. Wu H. Liu X. Terskikh A.V. Geschwind D.H. Kornblum H.I. J. Cell Biol. 2005; 170: 413-427Crossref PubMed Scopus (117) Google Scholar). At the functional level, MPK38 has been implicated to be involved in several cellular functions, including cell cycle, spliceosome assembly, gene expression, cell proliferation, carcinogenesis, and apoptosis (15Saito R. Tabata Y. Muto A. Arai K. Watanabe S. Mol. Cell Biol. 2005; 25: 6682-6693Crossref PubMed Scopus (19) Google Scholar, 16Beullens M. Vancauwenbergh S. Morrice N. Derua R. Ceulemans H. Waelkens E. Bollen M. J. Biol. Chem. 2005; 280: 40003-40011Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 17Jung H. Seong H.A. Ha H. J. Biol. Chem. 2008; 283: 34541-34553Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 18Gray D. Jubb A.M. Hogue D. Dowd P. Kljavin N. Yi S. Bai W. Frantz G. Zhang Z. Koeppen H. de Sauvage F.J. Davis D.P. Cancer Res. 2005; 65: 9751-9761Crossref PubMed Scopus (135) Google Scholar, 19Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, the exact biological function of MPK38 remains unclear.In this study, we show that there are direct physical and functional interactions between MPK38 and Smad proteins (Smad2, -3, -4, and -7), and that these interactions may play an important role in the regulation of Smad activities involved in TGF-β signaling.DISCUSSIONWe have previously shown that PDK1 interacts with intracellular signaling mediators of the TGF-β signaling pathway, such as Smad proteins (Smad2, -3, -4, and -7) and serine-threonine kinase receptor-associated protein, and that this interaction is involved in the reciprocal regulation of PI3K/PDK1 and TGF-β signaling (20Seong H.A. Jung H. Kim K.T. Ha H. J. Biol. Chem. 2007; 282: 12272-12289Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 30Seong H.A. Jung H. Choi H.S. Kim K.T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), suggesting a functional link between the PI3K/PDK1 and TGF-β signaling pathways. Furthermore, cross-talk between p53 and TGF-β signaling has been reported (31Cordenonsi M. Dupont S. Maretto S. Insinga A. Imbriano C. Piccolo S. Cell. 2003; 113: 301-314Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). These data reveal the possibility of cross-talk between cell-survival and death-inducing signals, or between cell death signaling pathways. We have recently found that MPK38 participates in ASK1 activation through direct interaction and phosphorylation, resulting in the stimulation of ASK1-mediated apoptosis (17Jung H. Seong H.A. Ha H. J. Biol. Chem. 2008; 283: 34541-34553Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These findings, together with TGF-β-induced stimulation of MPK38 kinase activity (Fig. 1A), raise the possibility that TGF-β signaling may be associated with MPK38-mediated signaling. Based on this, we speculated that the physical interaction between MPK38 and Smad proteins may occur in vivo.In this study, we demonstrated that MPK38 physically interacts with Smad proteins (Smad2, -3, -4, and -7) (Fig. 1). Moreover, our data indicate that the kinase activity of MPK38 is required for its ability to stimulate TGF-β-induced transcription (Fig. 5) and apoptosis (Fig. 9). These findings are similar to those from recent studies showing that PDK1 and its downstream target Akt physically interact with Smad2, -3, -4, and -7, and inhibit TGF-β signaling in a kinase-dependent manner (20Seong H.A. Jung H. Kim K.T. Ha H. J. Biol. Chem. 2007; 282: 12272-12289Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 32Song K. Wang H. Krebs T.L. Danielpour D. EMBO J. 2006; 25: 58-69Crossref PubMed Scopus (158) Google Scholar). In addition, in our study, wild-type MPK38 stimulated the normal movement of Smad proteins (Smad3 and -7) in response to TGF-β1, but the kinase-dead MPK38 did not show such an effect (Fig. 8). In this context, our current evidence indicates that MPK38 is a potential positive regulator of the TGF-β signaling pathway. This is in contrast to PDK1, which acts as a negative regulator of TGF-β signaling. However, our present results do not support the possibility that the MPK38-mediated stimulation of TGF-β signaling is due only to the physical interaction between MPK38 and Smad proteins, because the kinase-dead MPK38, which lacks the ability to regulate TGF-β signaling, was shown to associate with Smad proteins at levels similar to those of wild-type MPK38 (data not shown). These data suggest that MPK38-mediated phosphorylation of Smad proteins (Smad2, -3, -4, and -7) through direct interaction plays a crucial role in the positive regulation of TGF-β signaling induced by MPK38.Although C-terminal SXS phosphorylation of R-Smad by the type I TGF-β receptor is an important step in TGF-β signaling, additional phosphorylations by intracellular protein kinases are also involved in the regulation of Smad activities. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) and Rho-associated, coiled-coil containing protein kinase contribute to Smad3 phosphorylation at Ser204/208/213 in the linker region and stimulate its transcriptional activity (33Kamaraju A.K. Roberts A.B. J. Biol. Chem. 2005; 280: 1024-1036Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Similarly, our results showed that the MPK38-mediated phosphorylation of Smad3 at Ser204 plays a key role in the stimulation of TGF-β signaling (Fig. 6). However, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of Smad3 at Ser204 and other sites (Ser208 and Thr179), and of Smad2 at Ser245/250/255 and Thr220, showed an inhibitory effect on Smad3 activity (34Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (848) Google Scholar). These findings imply that MAPK-mediated phosphorylation of Smad2 and -3, in contrast to that of MPK38, appears to have a dual role in the regulation of Smad2 and -3. Growing evidence indicates that, in addition to these kinases, a variety of intracellular protein kinases (5Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar, 35Matsuura I. Denissova N.G. Wang G. He D. Long J. Liu F. Nature. 2004; 430: 226-231Crossref PubMed Scopus (413) Google Scholar, 36Engel M.E. McDonnell M.A. Law B.K. Moses H.L. J. Biol. Chem. 1999; 274: 37413-37420Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 37Mori S. Matsuzaki K. Yoshida K. Furukawa F. Tahashi Y. Yamagata H. Sekimoto G. Seki T. Matsui H. Nishizawa M. Fujisawa J. Okazaki K. Oncogene. 2004; 23: 7416-7429Crossref PubMed Scopus (168) Google Scholar, 38Wrighton K.H. Feng X.H. Cell Signal. 2008; 20: 1579-1591Crossref PubMed Scopus (41) Google Scholar), including cyclin-dependent kinase 2/4 (CDK2/4), c-Jun N-terminal kinase (JNK), G protein-coupled receptor kinase 2 (GRK2), and calmodulin-dependent kinase II, contribute to the phosphorylation of the Smad2 and -3 linker region, although the exact mechanism(s) by which phosphorylation in the Smad linker region regulates Smad-mediated signaling remains unclear. On the other hand, Smad4 is known to be constitutively phosphorylated in cells, although the phosphorylation sites are yet to be discovered (5Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar, 6Roelen B.A. Cohen O.S. Raychowdhury M.K. Chadee D.N. Zhang Y. Kyriakis J.M. Alessandrini A.A. Lin H.Y. Am. J. Physiol. Cell Physiol. 2003; 285: C823-C830Crossref PubMed Scopus (50) Google Scholar). In this regard, the discovery of phosphorylation of Smad4 at Ser343 by MPK38 (Fig. 3B), together with a recent result showing that ERK-mediated phosphorylation of Smad4 at Thr277 is involved in the nuclear translocation of Smad4 (6Roelen B.A. Cohen O.S. Raychowdhury M.K. Chadee D.N. Zhang Y. Kyriakis J.M. Alessandrini A.A. Lin H.Y. Am. J. Physiol. Cell Physiol. 2003; 285: C823-C830Crossref PubMed Scopus (50) Google Scholar), should aid in the understanding of TGF-β signal regulation. The inhibitory Smad proteins, Smad6 and Smad7, are phosphoproteins (7Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 8Pulaski L. Landström M. Heldin C.H. Souchelnytskyi S. J. Biol. Chem. 2001; 276: 14344-14349Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 39Glesne D. Huberman E. Oncogene. 2006; 25: 4086-4098Crossref PubMed Scopus (24) Google Scholar). It has been shown that protein kinase X, involved in macrophage differentiation, phosphorylates Smad6 at Ser435 (39Glesne D. Huberman E. Oncogene. 2006; 25: 4086-4098Crossref PubMed Scopus (24) Google Scholar). Our results showed that MPK38 is also involved in Smad6 phosphorylation at Thr176, and that this phosphorylation is important for the regulation of Smad6-mediated bone morphogenetic protein signaling (supplemental Fig. S3). In addition, Smad7 was found to be phosphorylated at Ser249 (8Pulaski L. Landström M. Heldin C.H. Souchelnytskyi S. J. Biol. Chem. 2001; 276: 14344-14349Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), although the intracellular kinases responsible for phosphorylating Smad7 have not yet been identified. Our results from this study have provided evidence that MPK38 stimulates TGF-β signaling by phosphorylating Smad7 at Thr96 (Fig. 6D), suggesting that MPK38 is a putative intracellular kinase for Smad7 phosphorylation in cells.We previously observed that PDK1-mediated inhibition of TGF-β signaling is accompanied by the of formation between the TGF-β receptor and Smad proteins (Smad3 and -7) (20Seong H.A. Jung H. Kim K.T. Ha H. J. Biol. Chem. 2007; 282: 12272-12289Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). the mechanism by which MPK38 stimulates TGF-β-induced transcriptional we also MPK38 modulate the association between the TGF-β receptor and Smad proteins (Smad3 and Our results showed that the ability of wild-type MPK38 to stimulate TGF-β-mediated transcriptional activation is on of to the TGF-β receptor (Fig. MPK38 increased formation between the TGF-β receptor and Smad3, and decreased formation between the TGF-β receptor and Smad7, to stimulation of TGF-β signaling. However, the association of TGF-β receptor with Smad proteins was not by the kinase-dead MPK38. These imply that the kinase activity of MPK38 is also required for of formation between the TGF-β receptor and Smad proteins, in with (Fig. and apoptosis (Fig. our findings suggest that MPK38 regulates TGF-β signaling through direct interaction and phosphorylation of Smad proteins, and also that Smad proteins may function as between TGF-β and MPK38-mediated signaling. The discovery of MPK38-mediated stimulation of TGF-β signaling should to TGF-β signaling is in MPK38 interacts and phosphorylates all of the Smad proteins (Smad2, -3, -4, and -7) we IntroductionType I transforming growth factor-β (TGF-β) receptor kinases are serine/threonine kinases with roles in a wide array of cellular processes. Phosphorylation of the C-terminal SXS motif in receptor-regulated Smads (R-Smads) by these kinases is a crucial step in TGF-β family signaling (1Abdollah S. Macías-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 2Macías-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 3Souchelnytskyi S. Tamaki K. Engström U. Wernstedt C. ten Dijke P. Heldin C.H. J. Biol. Chem. 1997; 272: 28107-28115Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). R-Smad phosphorylation results in the formation of heterodimeric complexes with the common Smad, Smad4. In addition, the phosphorylation of serine or threonine residues in the N-terminal or linker region has been observed in endogenous R-Smads (4Xu L. Biochim. Biophys. Acta. 2006; 1759: 503-513Crossref PubMed Scopus (77) Google Scholar). Several lines of evidence have demonstrated the existence of kinases responsible for N-terminal or linker region phosphorylation events, including mitogen-activated protein kinases (MAPKs), Ca2+ and calmodulin-dependent kinase II, cyclin-dependent kinase (CDK), 3The abbreviations used are: CDKcyclin-dependent kinaseSmadSma- and Mad-related proteinI-Smadinhibitory SmadMHMad homologyTβRtransforming growth factor-β receptorZPR9zinc-finger-like protein 9MPK38murine protein serine/threonine kinase 38PDK13-phosphoinositide-dependent protein kinase 1. protein kinase C (PKC), and G protein-coupled receptor kinase 2 (5Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar). Common Smad, Smad4, is constitutively phosphorylated in cells, although not all of the phosphorylation sites are known. Extracellular signal-regulated kinase (ERK) has been shown to phosphorylate Smad4 at Thr277, resulting in the prevention of nuclear translocation of Smad4 (6Roelen B.A. Cohen O.S. Raychowdhury M.K. Chadee D.N. Zhang Y. Kyriakis J.M. Alessandrini A.A. Lin H.Y. Am. J. Physiol. Cell Physiol. 2003; 285: C823-C830Crossref PubMed Scopus (50) Google Scholar). The inhibitory Smads (I-Smads), Smad6 and Smad7, are phosphorylated by uncharacterized kinases (7Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 8Pulaski L. Landström M. Heldin C.H. Souchelnytskyi S. J. Biol. Chem. 2001; 276: 14344-14349Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This provides a mechanism for integration of the Smad pathway with other signaling pathways, in which kinases act as essential mediators, which modulate Smad-mediated signaling. Thus, the identification and characterization of additional cellular kinases responsible for Smad phosphorylation could lead to a better understanding of the regulatory role of Smad phosphorylations, especially in the N-terminal and linker region, in TGF-β signaling.Murine protein serine/threonine kinase 38 (MPK38), also known as pEg3 kinase and maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase family of serine-threonine kinases (9Gil M. Yang Y. Lee Y. Choi I. Ha H. Gene. 1997; 195: 295-301Crossref PubMed Scopus (51) Google Scholar, 10Heyer B.S. Warsowe J. Solter D. Knowles B.B. Ackerman S.L. Mol. Reprod. Dev. 1997; 47: 148-156Crossref PubMed Google Scholar). The expression patterns of MPK38 during maturation of oocytes and pre-implantation development were found to be typical of that described for maternally expressed genes, including β-actin and E-cadherin (11Bachvarova R. Cohen E.M. De Leon V. Tokunaga K. Sakiyama S. Paynton B.V. Development. 1989; 106: 561-565Crossref PubMed Google Scholar, 12Ohsugi M. Hwang S.Y. Butz S. Knowles B.B. Solter D. Kemler R. Dev. Dyn. 1996; 206: 391-402Crossref PubMed Scopus (78) Google Scholar). This suggested that MPK38 may play a role in transducing signals in embryonic cells during pre-implantation, although its exact roles in development were not revealed. Additionally, MPK38 is also expressed in several other tissues, including embryonic stem cells (13Nakano I. Paucar A.A. Bajpai R. Dougherty J.D. Zewail A. Kelly T.K. Kim K.J. Ou J. Groszer M. Imura T. Freije W.A. Nelson S.F. Sofroniew M.V. Wu H. Liu X. Terskikh A.V. Geschwind D.H. Kornblum H.I. J. Cell Biol. 2005; 170: 413-427Crossref PubMed Scopus (117) Google Scholar), adult germ cells (14Heyer B.S. Kochanowski H. Solter D. Dev. Dyn. 1999; 215: 344-351Crossref PubMed Scopus (46) Google Scholar), hematopoietic stem cells (15Saito R. Tabata Y. Muto A. Arai K. Watanabe S. Mol. Cell Biol. 2005; 25: 6682-6693Crossref PubMed Scopus (19) Google Scholar), adult hematopoietic cells (9Gil M. Yang Y. Lee Y. Choi I. Ha H. Gene. 1997; 195: 295-301Crossref PubMed Scopus (51) Google Scholar), and neural stem cells (13Nakano I. Paucar A.A. Bajpai R. Dougherty J.D. Zewail A. Kelly T.K. Kim K.J. Ou J. Groszer M. Imura T. Freije W.A. Nelson S.F. Sofroniew M.V. Wu H. Liu X. Terskikh A.V. Geschwind D.H. Kornblum H.I. J. Cell Biol. 2005; 170: 413-427Crossref PubMed Scopus (117) Google Scholar). At the functional level, MPK38 has been implicated to be involved in several cellular functions, including cell cycle, spliceosome assembly, gene expression, cell proliferation, carcinogenesis, and apoptosis (15Saito R. Tabata Y. Muto A. Arai K. Watanabe S. Mol. Cell Biol. 2005; 25: 6682-6693Crossref PubMed Scopus (19) Google Scholar, 16Beullens M. Vancauwenbergh S. Morrice N. Derua R. Ceulemans H. Waelkens E. Bollen M. J. Biol. Chem. 2005; 280: 40003-40011Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 17Jung H. Seong H.A. Ha H. J. Biol. Chem. 2008; 283: 34541-34553Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 18Gray D. Jubb A.M. Hogue D. Dowd P. Kljavin N. Yi S. Bai W. Frantz G. Zhang Z. Koeppen H. de Sauvage F.J. Davis D.P. Cancer Res. 2005; 65: 9751-9761Crossref PubMed Scopus (135) Google Scholar, 19Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, the exact biological function of MPK38 remains unclear.In this study, we show that there are direct physical and functional interactions between MPK38 and Smad proteins (Smad2, -3, -4, and -7), and that these interactions may play an important role in the regulation of Smad activities involved in TGF-β signaling.