Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation
Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation
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DOI：
10.1074/jbc.m113.474593
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发表时间：
2013-06-07
影响因子：
4.8
通讯作者：
Weigert, Cora
中科院分区：
文献类型：
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作者：
Neukamm, Sabine S.;Ott, Jennifer;Weigert, Cora
胰岛素受体底物（IRS）2作为中间对接平台将胰岛素/IGF-1（胰岛素样生长因子1）信号转导至调节葡萄糖稳态、β细胞生长和存活的细胞内效应分子。以前，IRS 2已被鉴定为14-3-3相互作用蛋白。14-3-3蛋白可以通过位于不同基序内的磷酸化丝氨酸/苏氨酸残基结合其靶蛋白。在本研究中，在HEK 293细胞中证明了在用毛喉素或cAMP类似物8-（4-氯苯硫基）-cAMP刺激后14-3-3与IRS 2的结合。用PKA抑制剂H89或R-p-8-Br-cAMPS降低结合。在PKA共有基序上的IRS 2的磷酸化被毛喉素和PKA激活剂N-6-Phe-cAMP诱导，并被两种PKA抑制剂阻止。通过GST-14-3-3下拉分析，IRS 2上952位之后的氨基酸区域被鉴定为14-3-3结合区域。质谱分析显示丝氨酸1137和丝氨酸1138是cAMP依赖性的潜在PKA磷酸化位点。丝氨酸1137/1138突变为丙氨酸强烈降低cAMP依赖性14-3-3结合。放线菌酮的应用表明，毛喉素增强IRS 2蛋白在HEK 293细胞稳定表达IRS 2以及在原代肝细胞中的稳定性。刺激与毛喉素没有增加蛋白质的稳定性，无论是在14-3-3拮抗剂的存在下，或在双1137/1138丙氨酸突变体。因此，降低的IRS 2蛋白降解依赖于与14-3-3蛋白的相互作用和丝氨酸1137/1138的存在。我们提出丝氨酸1137/1138作为IRS 2上新的cAMP依赖性磷酸化位点，并显示其在14-3-3结合和IRS 2蛋白稳定性中的重要性。
Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, beta-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R-p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N-6-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.