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

Abstract

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.

Serine-Threonine Kinase Receptor-associated Protein Inhibits Apoptosis Signal-regulating Kinase 1 Function through Direct Interaction

“…Anti-PDK1, anti-phospho-AKT- (Thr 308 ), anti-AKT, anti-phospho-Bad (Ser 136 ), anti-Bad, and antiphospho-Ser/Thr antibodies were described previously (21,24). Anti-phospho-PDK1(Ser 241 ) antibody was from Cell Signaling Technology (Danvers, MA).…”

“…Small Interfering RNA (siRNA) Experiments-RNA interference experiments were performed as described previously (21). The STRAP-specific siRNA (number 515) of the oligonucleotides (5Ј-UUACGCAUAUAUGACUUGA-3Ј) targeting a coding region (aa 124 -129) on human STRAP (GenBank TM accession number BC000162) was used for transfection experiments.…”

“…STRAP reportedly acts as an activator of PDK1 (3-phosphoinositide-dependent protein kinase-1) by dissociating 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex (21). STRAP also contributes to tumor progression by blocking TGF-␤-mediated signaling, especially in colon and lung carcinomas, indicating the possible involvement of STRAP in tumorigenesis (20,22).…”

Signaling Receptors for TGF-β Family Members

“…STRAP has also been reported to link the kinase phosphoinositide-dependent kinase 1 (PDK1), which activates Akt, to TbRI and to promote its activation (Seong et al 2005), and to bind and inhibit the nucleoside diphosphate (NDP) kinase NM23-H1 (Seong et al 2007). STRAP thus affects TGF-b signaling via several mechanisms.…”

Oncogenic Serine-Threonine Kinase Receptor-Associated Protein Modulates the Function of Ewing Sarcoma Protein through a Novel Mechanism

“…These results suggest a broader role of STRAP in tumorigenesis and development. STRAP has also been shown to regulate the kinase activity of phosphoinositide-dependent kinase 1 through its physical interaction (21). A recent finding suggests how STRAP in survival motor neuron (SMN) protein complex plays an important role in pre-mRNA splicing (22).…”