Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR
Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR
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IGF-1、EGF、PMA 和 AICAR 对 AS160 的多位点磷酸化和 14-3-3 结合的调控
DOI：
10.1042/bj20070649
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发表时间：
2007-10-15
影响因子：
4.1
通讯作者：
MacKintosh, Carol
中科院分区：
文献类型：
--
作者：
Geraghty, Kathryn M.;Chen, Shuai;MacKintosh, Carol
AS 160（160 kDa 的 Akt 底物）介导胰岛素刺激的 GLUT4（葡萄糖转运蛋白 4）易位，但在缺乏 GLUT4 的胰岛素不敏感组织中广泛表达。通过14-3-3亲和层析分离AS160后，我们发现AS160与HEK（人胚胎肾）-293细胞中的14-3-3亚型的结合是由IGF-1（胰岛素样生长因子-1）、EGF（表皮生长因子）、PMA以及较小程度上的AICAR诱导的（5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核苷）。 AS 160-14-3-3 相互作用通过化学交联稳定并通过去磷酸化消除。 AS 160 上的 8 个残基（Ser(318)、Ser(341)、Thr(568)、Ser(570)、Ser(588)、Thr(642)、Ser(666) 和 Ser(751)）在 IGF-1、EGF、PMA 和 AICAR 的作用下发生差异磷酸化。 14-3-3 蛋白与 HA-AS160（其中 HA 是血凝素）的结合因 Thr(642) 突变而显着降低，并在 Thr642Ala/Ser341 Ala 双突变体中被消除。 AGC（蛋白激酶 A/蛋白激酶 G/蛋白激酶 C 家族）激酶 RSK1（p90 核糖体 S6 激酶 1）、SGK1（血清和糖皮质激素诱导的蛋白激酶 1）和 PKB（蛋白激酶 13）在体外显示出 AS160 磷酸化的不同特征：所有三种激酶均磷酸化 Ser（318）、Set（588）和 Thr（642）； RSK1 还磷酸化 Ser(341)、Ser(751) 以及较小程度的 Thr(568)； SGK1 磷酸化 Thr(568) 和 Ser(751)。 AMPK（AMP 激活蛋白激酶）优先磷酸化 Ser(588)，其他位点磷酸化较少。在细胞中，IGF-1 刺激的磷酸化和某些 EGF 刺激的磷酸化被 PI3K（磷酸肌醇 3-激酶）抑制剂抑制，而 RSK 抑制剂 BI-D1870 抑制 PMA 诱导的磷酸化。 HeLa 细胞中 LKB1 的表达以及 HEK-293 细胞中 AICAR 的使用促进了 Ser(588) 磷酸化，但仅促进了 Ser(341) 和 Thr(642) 磷酸化并与 14-3-3s 结合。然而矛盾的是，苯乙双胍激活 AMPK 却没有促进 AS 160 磷酸化。 IGF-1 诱导的新型磷酸化 Ser(666)-Pro 位点的磷酸化可被 AICAR 以及 TOS（mTOR 信号传导）样序列 (FEMDI) 和雷帕霉素的组合突变抑制。因此，尽管 AS160 是胰岛素、IGF-1、EGF、PMA 和 AICAR 的共同靶标，但这些刺激物会诱导独特的磷酸化模式和 14-3-3 结合，并由至少四种蛋白激酶介导。
AS 160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside). AS 160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS 160 (Ser(318), Ser(341), Thr(568), Ser(570), Ser(588), Thr(642), Ser(666) and Ser(751)) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr(642) and abolished in a Thr642Ala/Ser341 Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase 13) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser(318), Set(588) and Thr(642); RSK1 also phosphorylated Ser(341), Ser(751) and to a lesser extent Thr(568); and SGK1 phosphorylated Thr(568) and Ser(751). AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser(588), with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser(588), but only weak Ser(341) and Thr(642) phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS 160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser(666)-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.