The insulin-signaling network regulates blood glucose levels, controls metabolism, and when dysregulated, may lead to the development of type 2 diabetes. Although the role of tyrosine phosphorylation in this network is clear, only a limited number of insulin-induced tyrosine phosphorylation sites have been identified. To address this issue and establish temporal response, we have, for the first time, carried out an extensive, quantitative, mass spectrometrybased analysis of tyrosine phosphorylation in response to insulin. The study was performed with 3T3-L1 adipocytes stimulated with insulin for 0, 5, 15, and 45 min. It has resulted in the identification and relative temporal quantification of 122 tyrosine phosphorylation sites on 89 proteins. Insulin treatment caused a change of at least 1.3-fold in tyrosine phosphorylation on 89 of these sites. Among the responsive sites, 20 were previously known to be tyrosine phosphorylated with insulin treatment, including sites on the insulin receptor and insulin receptor substrate-1. The remaining 69 responsive sites have not previously been shown to be altered by insulin treatment. They were on proteins with a wide variety of functions, including components of the trafficking machinery for the insulin-responsive glucose transporter GLUT4. These results show that insulin-elicited tyrosine phosphorylation is extensive and implicate a number of hitherto unrecognized proteins in insulin action. Diabetes 55:2171-2179, 2006 M etabolic control is primarily regulated by the insulin-signaling network. In healthy individuals, insulin stimulates glucose uptake from the bloodstream into adipose tissue and skeletal muscle while inhibiting glucose production in the liver. Dysregulation of this network associated with insulin resistance causes an increase in blood glucose and lipid levels, often initially associated with an increase in insulin levels and eventually culminating in type 2 diabetes (1). Understanding the signaling network activated by insulin stimulation is crucial for identifying the causes and effects of network dysregulation and insulin resistance.Insulin binds to the insulin receptor at the cell surface and activates its tyrosine kinase activity, leading to autophosphorylation and phosphorylation of several receptor substrates. Phosphorylation of selected tyrosine sites on receptor substrates is known to activate different pathways leading to increased glucose uptake, lipogenesis, and glycogen and protein synthesis, as well as to stimulation of cell growth (1,2). In addition to activation of these pathways by tyrosine phosphorylation, several mechanisms of downregulating the response to insulin stimulation have also been identified. For instance, serine phosphorylation on insulin receptor substrate (IRS)-1 induced by a variety of factors has been shown to interfere with the activating effects of tyrosine phosphorylation by decreasing binding to the insulin receptor or increasing degradation of IRS-1 (1,3,4). Ser/Thr phosphorylation of the insulin receptor has also bee...

The mitogen-activated protein kinase (MAPK) ERK2 is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2,a form of ERK2 engineered to utilize an analog of ATP, to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, and mRNA processing, as well as regulation of the activity of the Rho-family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E-twenty six family of transcriptional regulators) was extensively phosphorylated on sites within canonical and non-canonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.

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