Global Assessment of Regulation of Phosphorylation of Insulin Receptor Substrate-1 by Insulin In Vivo in Human Muscle

Yi, Zhengping
Langlais, Paul
De Filippis, Elena A.
Luo, Moulun
Flynn, Charles R.
Schroeder, Stefanie
Weintraub, Susan T.
Mapes, Rebekka
Mandarino, Lawrence J.

From the ¹Center for Metabolic Biology, Arizona State University, Tempe, Arizona; the ²School of Life Sciences, Arizona State University, Tempe, Arizona; the ³Department of Kinesiology, Arizona State University, Tempe, Arizona; the ⁴Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas; and the ⁵Department of Physiology, The University of Texas Health Science Center, San Antonio, Texas.

Address correspondence and reprint requests to Lawrence J. Mandarino, PhD, Director, Center for Metabolic Biology, College of Liberal Arts and Sciences, P.O. Box 873704, Tempe, AZ 85287-3704. E-mail: [email protected]

Ang II, angiotensin II; CID, collision-induced dissociation; FTICR, Fourier transform ion cyclotron resonance; hIRS, human insulin receptor substrate; HPLC, high-performance liquid chromatography; IRS, insulin receptor substrate; PI, phosphatidylinositol; PKC, protein kinase C; PTB, phosphotyrosine binding

Published ahead of print at http://diabetes.diabetesjournals.org on 15 March 2007. DOI: 10.2337/db06-1355.

Z.Y., P.L., E.A.D., and M.L. contributed equally to this work.

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-1355.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication 22 September 2006 and accepted in revised form 8 March 2007

Diabetes 56(6):p 1508-1516, June 2007.

OBJECTIVE

Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans.

RESEARCH DESIGN AND METHODS

In this study, we describe our use of capillary high-performance liquid chromotography electrospray tandem mass spectrometry to identify/quantify site-specific phosphorylation of IRS-1 in human vastus lateralis muscle obtained by needle biopsy basally and after insulin infusion in four healthy volunteers.

RESULTS

Twenty-two serine/threonine phosphorylation sites were identified; 15 were quantified. Three sites had not been previously identified (Thr⁴⁹⁵, Ser⁵²⁷, and S¹⁰⁰⁵). Insulin increased the phosphorylation of Ser³¹², Ser⁶¹⁶, Ser⁶³⁶, Ser⁸⁹², Ser¹¹⁰¹, and Ser¹²²³ (2.6 ± 0.4, 2.9 ± 0.8, 2.1 ± 0.3, 1.6 ± 0.1, 1.3 ± 0.1, and 1.3 ± 0.1–fold, respectively, compared with basal; P < 0.05); phosphorylation of Ser³⁴⁸, Thr⁴⁴⁶, Thr⁴⁹⁵, and Ser¹⁰⁰⁵ decreased (0.4 ± 0.1, 0.2 ± 0.1, 0.1 ± 0.1, and 0.3 ± 0.2–fold, respectively; P < 0.05).

CONCLUSIONS

These results provide an assessment of IRS-1 phosphorylation in vivo and show that insulin has profound effects on IRS-1 serine/threonine phosphorylation in healthy humans.