Regulation of transforming growth factor-beta 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-beta (TGF-beta) 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-beta 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-beta signaling with STRAP by contributing to the stable association between TGF-beta receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-beta. 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-beta signaling pathways.
Seong
Hyun-A
HA
Department of Biochemistry, Research Center for Bioresource and Health, Biotechnology Research Institute, School of Life Sciences, Chungbuk National University, Cheongju 361-763. hyunha@chungbuk.ac.kr
Jung
Haiyoung
H
Choi
Hueng-Sik
HS
Kim
Kyong-Tai
KT
Ha
Hyunjung
H
D064413:3-Phosphoinositide-Dependent Protein Kinases; D021581:Active Transport, Cell Nucleus; D000730:Androstadienes / Q000494:pharmacology; D017209:Apoptosis; D020134:Catalytic Domain; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000737:chemistry*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism; D051906:Smad7 Protein / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D007328:Insulin; D009363:Neoplasm Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C403447:STRAP protein, human; D051900:Smad3 Protein; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin