This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXXS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.Protein phosphorylation is a key cellular regulatory mechanism. The human genome contains ϳ1000 kinases. A major issue is to identify the protein substrates for each kinase. A number of different approaches have been developed (reviewed in Ref. 1). These include: in vitro phosphorylation of cell homogenates with recombinant kinases, screening of expression libraries with recombinant kinases, searching for kinase interacting proteins by the yeast two-hybrid screen, and the generation of mutated kinases that can function only with an ATP derivative. Each of these methods has its advantages and limitations, and additional methods are needed.Each serine kinase typically phosphorylates Ser/Thr within a particular motif, and for many kinases the motif has been defined through identification of the sites of phosphorylation on substrate proteins and on peptide libraries (2). Phosphospecific antibodies against the phosphorylated form of these motifs are available or can be generated. Thus, immunoprecipitation with these antibodies, when combined with tandem mass spectrometry of tryptic peptides, offers an approach for the isolation and identification of substrates for specific serine kinases. In the past this approach has been used for the identification of substrates for tyrosine kinases through the use of antibodies against phosphotyrosine, but to our knowledge it has not previously been used with serine kinases.In the present study, we applied this method to find substrates for the protein kinase Akt (also known as protein kinase B), which specifically phosphorylates Ser or Thr in the motif RXRXXS/T (3). We have employed 3T3-L1 adipocytes, a cell type in which Akt is rapidly activated by insulin treatment (4). This method has led to the isolation of a novel target for Akt that contains a Rab GAP 1 domain and two PTB domains. EXPERIMENTAL PROCEDURESAntibodies-A key reagent for this study is the antibody for the Akt phosphomotif RXRXXpS/T, where X is any amino acid...

The insulin receptor substrates (IRS) 1 and 2 are required for normal growth and glucose homeostasis in mice. To determine whether IRS-3, a recently cloned member of the IRS family, is also involved in the regulation of these, we have generated mice with a targeted disruption of the IRS-3 gene and characterized them. Compared with wild-type mice, the IRS-3-null mice showed normal body weight throughout development, normal blood glucose levels in the fed and fasted state and following an oral glucose bolus, and normal fed and fasted plasma insulin levels. IRS-3 is most abundant in adipocytes and is tyrosine-phosphorylated in response to insulin in these cells. Therefore, isolated adipocytes were analyzed for changes in insulin effects. Insulinstimulated glucose transport in the adipocytes from the IRS-3-null mice was the same as in wild-type cells. The extent of tyrosine phosphorylation of IRS-1/2 following insulin stimulation was similar in adipocytes from IRS-3-null and wild-type mice, and the insulin-induced association of tyrosine-phosphorylated IRS-1/2 with phosphatidylinositol 3-kinase and SHP-2 was not detectably increased by IRS-3 deficiency. Thus, IRS-3 was not essential for normal growth, glucose homeostasis, and glucose transport in adipocytes, and in its absence no significant compensatory augmentation of insulin signaling through IRS-1/2 was evident.The IRSs 1 play a key role in signal transduction from the insulin receptor (reviewed in Ref. 1). They are the major intracellular targets for phosphorylation by the activated insulin receptor tyrosine kinase. In addition, they are also substrates for the insulin-like growth factor I receptor and for tyrosine kinases associated with the receptors for growth hormone and some interleukins and interferons. Four members of the IRS family (IRS-1, -2, -3, and -4) are now known. Each IRS contains at its N terminus a pleckstrin homology domain and a phosphotyrosine binding domain, both of which are required for efficient phosphorylation by the insulin receptor tyrosine kinase. The large C-terminal portion of each IRS contains many tyrosine phosphorylation sites in motifs that can associate with SH2 domain-containing proteins, notably PI 3-kinase, the adaptor protein Grb-2 (complexed with Sos, the guanine nucleotide exchange factor for Ras), and the tyrosine phosphatase SHP-2. The interaction of the tyrosine-phosphorylated IRSs with PI 3-kinase and Grb-2/Sos stimulates their activities, leading to the elevation of PI 3,4,5-trisphosphate and the GTP form of Ras. These in turn activate protein kinase cascades, resulting in the well established cellular effects of insulin, including the stimulation of glucose transport, glycogen synthesis, and protein synthesis and alterations in gene transcription (reviewed in Refs. 1 and 2).The occurrence of four members of the IRS family raises the question of the physiological roles of each. Targeted disruption of the IRS genes provides an approach to address this question. To date mice lacking IRS-1 and IRS-2 have been described...

The insulin receptor substrates (IRSs) are key components in signaling from the insulin receptor, and consequently any proteins that interact with them are expected to participate in insulin signaling. In this study we have searched for proteins that interact with IRS-4 by identifying the proteins that coimmunoprecipitated with IRS-4 from human embryonic kidney 293 cells by microsequencing through mass spectrometry. A group of proteins was found. These included phosphatidylinositol 3-kinase, a protein previously identified as an IRS-4 interactor, and several proteins for which there was no previous evidence of IRS-4 association. One of these proteins, named IRAS, that had been found earlier in another context was examined in detail. The results from the overexpression of IRAS, where its amount was about the same as that of IRS-4, indicated that IRAS associated directly with IRS-4 and showed that the increased complexation of IRS-4 with IRAS did not alter the insulin-stimulated tyrosine phosphorylation of IRS-4 or the association of IRS-4 with phosphatidylinositol 3-kinase or Grb2. On the other hand, overexpression of IRAS enhanced IRS-4-dependent insulin stimulation of the extracellularly regulated kinase. The domains of IRAS and IRS-4 responsible for the association of these two proteins were identified, and it was shown that IRAS also associates with IRS-1, IRS-2, and IRS-3.The insulin receptor substrates (IRS)-1, 1 IRS-2, IRS-3, and IRS-4 are a family of four similar proteins that play a key role in signaling from the insulin receptor (reviewed in Ref. 1). The activated insulin receptor phosphorylates each IRS on multiple tyrosine residues. The tyrosine-phosphorylated form of the IRS then binds to various SH2 domain-containing signaling proteins. These include the lipid kinase PI 3-kinase and the linker protein Grb2, which is complexed with Sos, the guanine nucleotide exchanger for Ras. Association of the IRS with PI 3-kinase stimulates its activity, and the resulting elevation of the lipid PI 3,4,5-trisphosphate leads to activation of Akt kinases. Association of the IRS with the Grb2-Sos complex enhances guanine nucleotide exchange on Ras, and the resulting elevation of the GTP form of Ras leads to activation of ERK kinases. The stimulation of PI 3-kinase is a required part of the signal transduction pathway to many of the cellular effects of insulin (2). The architecture of each IRS consists of an amino-terminal PH and PTB domain and a large carboxyl-terminal region with the sites of tyrosine phosphorylation. The PTB domain binds directly to the activated insulin receptor, and both the PH and PTB domains are required for efficient tyrosine phosphorylation of the IRS.In an effort to identify additional proteins involved in insulin signaling, we and others have searched for proteins that associate with the IRSs. To date three approaches have been taken. First, proteins that were expected to interact with an IRS for various reasons have been selected, and their association with the IRS has been examined, gener...

No abstract