Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

Kathryn M. Geraghty, Shuai Chen, Jean E. Harthill, Adel F. Ibrahim, Rachel Toth, Nick A. Morrice, Franck Vandermoere, Greg B. Moorhead, D. Grahame Hardie, Carol MacKintosh

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    128 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)231-241
    Number of pages11
    JournalBiochemical Journal
    Volume407
    Early online date6 Jul 2007
    DOIs
    Publication statusPublished - 15 Oct 2007

    Keywords

    • 14-3-3
    • Akt/protein kinase B (PKB)
    • Akt substrate of 160 kDa (AS160)
    • GTPase-activating protein (GAP)
    • p90 ribosomal S6 kinase (RSK)
    • Serum- and glucocorticoid-induced protein kinase (SGK)

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