title: Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.
abstract: AS160 (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-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) 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 Thr642 and abolished in a Thr642Ala/Ser341Ala 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 B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, 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 Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-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.
authors.affiliation: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Geraghty
authors.forename: Shuai
authors.initials: S
authors.lastname: Chen
authors.forename: Jean E
authors.initials: JE
authors.lastname: Harthill
authors.forename: Adel F
authors.initials: AF
authors.lastname: Ibrahim
authors.forename: Rachel
authors.initials: R
authors.lastname: Toth
authors.forename: Nick A
authors.initials: NA
authors.lastname: Morrice
authors.forename: Franck
authors.initials: F
authors.lastname: Vandermoere
authors.forename: Greg B
authors.initials: GB
authors.lastname: Moorhead
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
authors.forename: Carol
authors.initials: C
authors.lastname: MacKintosh
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000596:Amino Acids; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D012265:Ribonucleotides / Q000494:pharmacology*
chemical_list: D048948:14-3-3 Proteins; D000596:Amino Acids; D020690:GTPase-Activating Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D012265:Ribonucleotides; C518767:TBC1D4 protein, human; D000620:Aminoimidazole Carboxamide; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; C031143:AICA ribonucleotide