Insulin resistance is a characteristic feature of obesity and type 2 diabetes mellitus. Briefly, the condition is characterized by defects at many levels, with decreases in insulin receptor concentration, phosphorylation of IRS-1 and 2, PI 3-kinase (PI3K) activity, AKT activity, glucose transporter translocation, and the activity of intracellular enzymes. Single defect in the insulin-signaling pathways such as knockout of the IR, IRS-2 or AKT2, can produce diabetes, whereas, knockout of the p85 subunit of PI3K, AKT1, IRS-1 or GLUT4 does not. PKClambda knockout mouse embryonic stem cells show severe impairment of insulin's effect on glucose transport, whereas, PKClambda gene knockout mice are embryonic lethal. Both, AKT2 and PKClambda, serving downstream of PI3K are activated by multiple agents via diverse initial signaling pathways. They are required for multiple insulin-stimulated effects such as: glucose transport, glycolysis, protein synthesis, lipogenesis, glycogen synthesis, suppression of gluconeogenesis, cell survival, and proliferation. We hypothesize that, regardless of initial mechanism, activated AKT2 and PKClambda are implicated in multiple, diverse, and sometimes opposing effects. For the families of these differentially regulated kinases with distinct physiological functions, the number of substrates identified to date mediating their diverse roles are surprisingly limited. We propose to identify their novel downstream substrates/targets that mediate the diverse functions in normal physiology and in insulin resistance. Since protein kinases contain similar ATP binding domains we will test this hypothesis by, (1) generating analog sensitive ATP bonding pocket mutants of AKT2 and PKClambda and identify compatible, highest affinity synthetic ATP analogs in invitro kinase assays, (2) Adenovirally overexpress these mutants in 3T3-L1 adipocytes, perform kinase reactions in the presence of radiolabelled synthetic ATP analogs, and identify novel, insulin-regulated substrates. (3) characterize candidate substrates identified in specific Aim # 2. In summary, we propose to utilize a novel technique of using analog sensitive mutants and modified ATP analogs to identify novel substrates of AKT2 and aPKCs, in insulin target cells. We believe that the findings from the proposed investigations will improve our general understanding of players of insulin resistance and offer potential insight into new therapeutic modalities.
| Sharma, Prem M; Son, Hyun-Shik; Ugi, Satoshi et al. (2005) Mechanism of SHIP-mediated inhibition of insulin- and platelet-derived growth factor-stimulated mitogen-activated protein kinase activity in 3T3-L1 adipocytes. Mol Endocrinol 19:421-30 |
