PDK1 Recruitment to the SHPS-1 Signaling Complex Enhances Insulin-like Growth Factor-I-stimulated AKT Activation and Vascular Smooth Muscle Cell Survival

Abstract

In vascular smooth muscle cells, exposed to hyperglycemia and insulin-like growth factor-I (IGF-I), SHPS-1 functions as a scaffold protein, and a signaling complex is assembled that leads to AKT activation. However, the underlying mechanism by which formation of this complex activates the kinase that phosphorylates AKT (Thr 308 ) is unknown. Therefore, we investigated the mechanism of PDK1 recruitment to the SHPS-1 signaling complex and the consequences of disrupting PDK1 recruitment for downstream signaling. Our results show that following IGF-I stimulation, PDK1 is recruited to SHPS-1, and its recruitment is mediated by Grb2, which associates with SHPS-1 via its interaction with Pyk2, a component of the SHPS-1-associated complex. A proline-rich sequence in PDK1 bound to an Src homology 3 domain in Grb2 in response to IGF-I. Disruption of Grb2-PDK1 by expression of either a Grb2 Src homology 3 domain or a PDK1 proline to alanine mutant inhibited PDK1 recruitment to SHPS-1, leading to impaired IGF-Istimulated AKT Thr 308 phosphorylation. Following its recruitment to SHPS-1, PDK1 was further activated via Tyr 373/376 phosphorylation, and this was required for a maximal increase in PDK1 kinase activity and AKT-mediated FOXO3a Thr 32 phosphorylation. PDK1 recruitment was also required for IGF-I to prevent apoptosis that occurred in response to hyperglycemia. Assembly of the Grb2-PDK1 complex on SHPS-1 was specific for IGF-I signaling because inhibiting PDK1 recruitment to SHPS-1 had no effect on EGF-stimulated AKT Thr 308 phosphorylation. These findings reveal a novel mechanism for recruitment of PDK1 to the SHPS-1 signaling complex, which is required for IGF-I-stimulated AKT Thr 308 phosphorylation and inhibition of apoptosis.AKT is phosphorylated at two key regulatory sites, Thr 308 in the activation loop of the catalytic domain and Ser 473 in the COOH-terminal domain. PDK1 (3-phosphoinositidedependent kinase 1) is the kinase responsible for phosphorylation of Thr 308 (1), and this is required for AKT-mediated inhibition of apoptosis (2-5). Although constitutive autophosphorylation of PDK1 Ser 241 is required for PDK1 kinase activity, it is not regulated by growth factors (1, 6). However, PDK1 activity is regulated by changes in its conformation, tyrosine phosphorylation, and subcellular localization (7-12). PDK1 undergoes tyrosine phosphorylation in response to insulin, angiotensin II, high glucose, and insulin-like growth factor-I (IGF-I) 2 (9,(11)(12)(13)(14)(15)(16)(17). Previous studies indicate that optimal activation of PDK1 requires phosphorylation of Tyr 373/376 (11,12,14,17), and growth factor receptor activation leads to PDK1 recruitment to the plasma membrane, followed by sequential phosphorylation of Tyr 9 and then Tyr 373/376 (14, 17). A previous study suggested that Pyk2 (proline-rich tyrosine kinase 2) plays an important role in PDK1 activation. Tanniyama et al. (9) showed that Pyk2 was required for optimal PDK1 tyrosine phosphorylation in response to angiotensin II and that Pyk2 and PDK1 were co...

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