Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.
Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-terminal regions of IRS-1 treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo.
Yi
Zhengping
Z
School of Life Sciences, Department of Kinesiology, Arizona State University, Tempe, Arizona 85287, USA.
Luo
Moulun
M
Carroll
Christopher A
CA
Weintraub
Susan T
ST
Mandarino
Lawrence J
LJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1