Phosphorylation of the CREB-specific coactivator TORC2 at Ser(307) regulates its intracellular localization in COS-7 cells and in the mouse liver.
Phosphorylation of the CREB-specific coactivator TORC2 at Ser(307) regulates its intracellular localization in COS-7 cells and in the mouse liver.
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DOI：
10.1152/ajpendo.00525.2009
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发表时间：
2010-09
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影响因子：
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通讯作者：
T. Uebi;Mitsuhiro Tamura;N. Horike;Y. Hashimoto;H. Takemori
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作者：
T. Uebi;Mitsuhiro Tamura;N. Horike;Y. Hashimoto;H. Takemori
CREB特异性共激活因子TORC 2（也称为CRTC 2）上调肝脏中的致瘤基因表达。盐诱导激酶（SIK）家族酶通过磷酸化抑制TORC 2并将其定位在细胞质中。发现Ser（171）和Ser（275）在胰腺β细胞中被磷酸化。钙调神经磷酸酶（Cn）被认为是Ser（275）磷酸酶，因为它的抑制剂环孢菌素A（CsA）稳定磷酸-Ser（275）并将TORC 2保留在细胞质中。由于Ser（171）去磷酸化的调节尚未完全阐明，我们用一系列剂量的冈田酸（OA）（PP 2A/PP 1的抑制剂）和各种磷酸酶的过表达进行了实验，发现PP 1作为TORC 2的激活剂发挥作用，而PP 2A作为抑制剂发挥作用。在使用TORC 2突变体的进一步研究中，我们检测到TORC 2的细胞内分布和转录活性之间的分离。另外的突变体分析表明存在第三个磷酸化位点Ser（307）。在COS-7细胞中，Ser（307）破坏的TORC 2组成性定位于细胞核中，但其共激活因子活性通常被SIK 1抑制。CsA而非OA稳定了Ser（307）的磷酸基团，表明Ser（171）和Ser（307）的差异去磷酸化协同调节TORC 2活性，并且TORC 2的核定位不足以作为共激活剂发挥作用。因为COS-7细胞系可能不具有促凋亡程序的信号级联，我们接下来检查了Ser（307）和Ser（171）对小鼠肝脏中TORC 2功能的重要性。Ser（171）和Ser（307）的磷酸化水平随着小鼠肝脏的禁食或进食条件和胰岛素抵抗而改变，这些情况通过CsA/OA处理和PP 1/PP 2A/Cn的过表达而改变。这些结果表明，多个磷酸化位点及其磷酸酶可能在调节TORC 2/CREB介导的肝脏致凋亡程序中发挥重要作用。
The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. Ser(171) and Ser(275) were found to be phosphorylated in pancreatic beta-cells. Calcineurin (Cn) is proposed as the Ser(275) phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-Ser(275) and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at Ser(171) has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, Ser(307). The Ser(307)-disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at Ser(307), suggesting that differential dephosphorylation at Ser(171) and Ser(307) cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of Ser(307) and Ser(171) for TORC2's function in mouse liver. Levels of phosphorylation at Ser(171) and Ser(307) changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver.