Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR
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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-β-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.
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The S595 site is a potential AMPK site.
“Worth mentioning is that the S595 site has also been shown to be a potential AMPK site [80], and indeed, its phosphorylation was increased upon in situ contraction in muscle extracts from both wild-type and TBC1D4 T649A KI mice [78].”
From AMPK and Exercise: Glucose Uptake and Insulin Sensitivity (2013) · cites this paper as [80]
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- Authors
- Geraghty, K. M., Chen, S., Harthill, J. E., Ibrahim, A. F., Toth, R., Morrice, N. A., Vandermoere, F., Moorhead, G. B., Hardie, D. G., & MacKintosh, C.
- Affiliations
- University of Dundee (GB)
- University of Calgary (CA)
- Corresponding author
- Chen, Shuai
- Toth, Rachel
- Vandermoere, Franck
- MacKintosh, Carol
- ORCID
- Venue
- Biochemical Journal
- Publisher
- Portland Press Ltd.
- Published
- 2007-09-25
- Volume
- 407
- Issue
- 2
- Pages
- 231-241
- ISSN
- 0264-6021 (ISSN-L)
- 1470-8728
- PMC
- PMC2049023
- OpenAlex
- W2052511418
- Cited by
- 181
- References
- 49
- Citations by year
- 2026: 1 · 2025: 7 · 2024: 3 · 2023: 5 · 2022: 9 · 2021: 3 · 2020: 7 · 2019: 8 · 2018: 7 · 2017: 5 · 2016: 7 · 2015: 8 · 2014: 14 · 2013: 13 · 2012: 16
- FWCI
- 6.63
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Biochemical Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Discovery Research Portal (University of Dundee) (publishedVersion)
- HAL (submittedVersion, other-oa)
- Trinity's Access to Research Output (TARA) (Trinity College Dublin) (submittedVersion, other-oa)
- Funders
- Pfizer
- AstraZeneca
- GlaxoSmithKline
- University of Dundee
- Merck KGaA
- Diabetes UK
- European Commission (LSHMCT-2004-005272)
- Medical Research Council
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › 14-3-3 protein interactions
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Keywords
- AS160, 14-3-3 protein binding, multisite phosphorylation, insulin-like growth factor I, epidermal growth factor, PMA, AICAR, AMPK, phosphatidylinositol 3-kinase, RSK1, SGK1, Akt, GLUT4 translocation, insulin resistance
- MeSH terms
- Amino Acids, Aminoimidazole Carboxamide, Binding Sites, Cell Line, Epidermal Growth Factor, Humans, Hypoglycemic Agents, Insulin, Insulin-Like Growth Factor I, Phosphorylation, Protein Binding, Ribonucleotides, GTPase-Activating Proteins, 14-3-3 Proteins
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