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Regulation of novel sites on AS160 by insulin and AICAR in human skeletal muscle
Author(s) -
Sharoff Carrie Grace,
Langlais Paul,
Yi ZhengPing,
Mandarino Lawrence J.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.783.4
Subject(s) - phosphorylation , skeletal muscle , insulin , chemistry , medicine , threonine , endocrinology , protein kinase b , insulin receptor , serine , biochemistry , biology , insulin resistance
AS160 is an important mediator of both insulin and contraction‐stimulated glucose uptake in skeletal muscle. Phosphorylation by upstream kinases at several serine and threonine residues directly affects the function of this protein; however, the knowledge of this is limited to sites where phospho‐specific antibodies are available. To more effectively study the effects of Akt and AMPK on AS160 phosphorylation, we employed HPLC‐electrospray ionization hybrid (LTQ‐FT/ICR) tandem mass spectrometry techniques to identify additional serine and threonine residues that are phosphorylated in AS160. CHO/IR cells were transiently transfected with cDNA encoding full length wild‐type human AS160. 24 hours after transfection, serum starved cells were treated with insulin (n=2, 100nM, t=15min) or AICAR (n=3, 2mM, t=60 min). Top 10 data‐dependent HPLC‐ESI‐MS/MS analysis confirmed the identity of 18 phosphorylation sites, 13 of which are previously unidentified. AICAR increased pSer725 by 33±0.3% while insulin increased pSer787 by 46±0.2% (p<0.05) in these novel sites. Next, we assessed the effect of insulin on pSer787 in vivo in human skeletal muscle. Basal and 2‐hr insulin stimulated muscle biopsy samples from lean individuals were analyzed. Insulin increased pSer787 by 60%. These data show that insulin and AICAR independently regulate phosphorylation at Ser787 and Ser725, respectively.