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BIOLOGICAL SCIENCES / CELL BIOLOGY
Dissection of the insulin signaling pathway via quantitative phosphoproteomics
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*Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; Department of Biochemistry and Molecular Biology, Center for Experimental BioInformatics, University of Southern Denmark, 5230 Odense, Denmark; and Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215
Contributed by C. Ronald Kahn, December 21, 2007 (received for review November 27, 2007)
The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full spectrum of the tyrosine phosphorylation cascade, we have defined the tyrosine-phosphoproteome of the insulin signaling pathway, using high resolution mass spectrometry in combination with phosphotyrosine immunoprecipitation and stable isotope labeling by amino acids in cell culture (SILAC) in differentiated brown adipocytes. Of 40 identified insulin-induced effectors, 7 have not previously been described in insulin signaling, including SDR, PKC
binding protein, LRP-6, and PISP/PDZK11, a potential calcium ATPase binding protein. A proteomic interaction screen with PISP/PDZK11 identified the calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells.
diabetes | insulin action | tyrosine phosphorylation
Author contributions: M.K., I.K., B.B., Y.-H.T., C.R.K., and M.M. designed research; M.K. and Y.-H.T. performed research; Y.-H.T. and C.R.K. contributed new reagents/analytic tools; M.K., I.K., B.B., Y.-H.T., and M.M. analyzed data; and M.K., C.R.K., and M.M. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0711713105/DC1.
To whom correspondence may be addressed. E-mail: c.ronald.kahn{at}joslin.harvard.edu or mmann{at}biochem.mpg.de
© 2008 by The National Academy of Sciences of the USA
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