Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: Regulatory role of serine 1223

Moulun Luo, Sara Reyna, Lishan Wang, ZhengPing Yi, Christopher Carroll, Lily Q. Dong, Paul R Langlais, Susan T. Weintraub, Lawrence J. Mandarino

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser 1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser 1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser 1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser 1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.

Original languageEnglish (US)
Pages (from-to)4410-4416
Number of pages7
JournalEndocrinology
Volume146
Issue number10
DOIs
StatePublished - Oct 2005
Externally publishedYes

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Insulin Receptor Substrate Proteins
Threonine
Serine
Mass Spectrometry
Phosphorylation
Insulin
Phosphoric Monoester Hydrolases
Tyrosine
SH2 Domain-Containing Protein Tyrosine Phosphatases
Phosphatidylinositol 3-Kinase
Mutation
Protein Tyrosine Phosphatases
Electrospray Ionization Mass Spectrometry
CHO Cells
Insulin Receptor
Tandem Mass Spectrometry
Proline
Mutagenesis
Alanine
Sequence Analysis

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis : Regulatory role of serine 1223. / Luo, Moulun; Reyna, Sara; Wang, Lishan; Yi, ZhengPing; Carroll, Christopher; Dong, Lily Q.; Langlais, Paul R; Weintraub, Susan T.; Mandarino, Lawrence J.

In: Endocrinology, Vol. 146, No. 10, 10.2005, p. 4410-4416.

Research output: Contribution to journalArticle

Luo, M, Reyna, S, Wang, L, Yi, Z, Carroll, C, Dong, LQ, Langlais, PR, Weintraub, ST & Mandarino, LJ 2005, 'Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: Regulatory role of serine 1223', Endocrinology, vol. 146, no. 10, pp. 4410-4416. https://doi.org/10.1210/en.2005-0260
Luo, Moulun ; Reyna, Sara ; Wang, Lishan ; Yi, ZhengPing ; Carroll, Christopher ; Dong, Lily Q. ; Langlais, Paul R ; Weintraub, Susan T. ; Mandarino, Lawrence J. / Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis : Regulatory role of serine 1223. In: Endocrinology. 2005 ; Vol. 146, No. 10. pp. 4410-4416.
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