PDK1 Recruitment to the SHPS-1 Signaling Complex Enhances Insulin-like Growth Factor-I-stimulated AKT Activation and Vascular Smooth Muscle Cell Survival
PDK1 Recruitment to the SHPS-1 Signaling Complex Enhances Insulin-like Growth Factor-I-stimulated AKT Activation and Vascular Smooth Muscle Cell Survival
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DOI：
10.1074/jbc.m110.155325
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发表时间：
2010-09-17
影响因子：
4.8
通讯作者：
Clemmons, David R.
中科院分区：
文献类型：
--
作者：
Shen, Xinchun;Xi, Gang;Clemmons, David R.
在暴露于高血糖和胰岛素样生长因子-I（IGF-I）的血管平滑肌细胞中，SHPS-1作为支架蛋白发挥作用，并且组装导致AKT活化的信号复合物。然而，这种复合物的形成激活磷酸化AKT（Thr（308））的激酶的潜在机制尚不清楚。因此，我们研究了PDK 1募集到SHPS-1信号复合物的机制以及破坏PDK 1募集到下游信号的后果。我们的研究结果表明，IGF-I刺激后，PDK 1被招募到SHPS-1，其招募是由Grb 2介导的，Grb 2通过与Pyk 2（SHPS-1相关复合物的一个组分）的相互作用与SHPS-1相关。PDK 1中富含脯氨酸的序列与Grb 2中的Src同源性3结构域结合，以响应IGF-I。通过表达Grb 2 Src同源性3结构域或PDK 1脯氨酸-丙氨酸突变体破坏Grb 2-PDK 1可抑制PDK 1向SHPS-1的募集，导致IGF-I刺激的AKT Thr（308）磷酸化受损。在其募集到SHPS-1后，PDK 1通过Tyr（373/376）磷酸化进一步活化，这是PDK 1激酶活性和AKT介导的FOXO 3a Thr（32）磷酸化最大增加所需的。PDK 1的募集也是IGF-I防止高血糖引起的细胞凋亡所必需的。Grb 2-PDK 1复合物在SHPS-1上的组装对IGF-I信号传导具有特异性，因为抑制PDK 1募集至SHPS-1对EGF刺激的AKT Thr（308）磷酸化没有影响。这些发现揭示了PDK 1募集到SHPS-1信号复合物的新机制，这是IGF-I刺激的AKT Thr（308）磷酸化和抑制凋亡所必需的。
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-I-stimulated 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.