IRS-1 tyrosine phosphorylation reflects insulin-induced metabolic and mitogenic responses in 3T3-L1 pre-adipocytes.
We determined the involvement of Tyr-1158 within the regulatory loop of the insulin receptor (IR) in the generation of insulin-specific responses in situ. For this purpose chimeric receptors with an epidermal growth factor (EGF) receptor extracellular domain and an IR cytoplasmic domain (EIR) were constructed, which allow activation of the cytoplasmic IR domain without activation of endogenous wt-IRs. Tyr-1158 of the chimera EIR was exchanged for Phe, creating a mutant chimeric receptor (EIR-Y1158F). Chimeric receptors were expressed in 3T3-L1 pre-adipocytes, which do not show insulin-specific responses upon EGF stimulation. We found that pre-adipocytes expressing EIR-Y1158F were impaired in their ability to stimulate glycogen synthesis and DNA synthesis upon maximal stimulation with EGF. EIR-Y1158F was impaired in its ability to phosphorylate insulin receptor substrate (IRS)-1 and induce downstream signals of IRS-1 phosphorylation, such as the association of IRS-1 with phosphatidyl-inositol-3'-kinase and the activation of protein kinase B (Akt). In contrast with the phosphorylation of IRS-1, the phosphorylation of IRS-2 and extracellular regulated protein kinase-1/-2 was normal in EIR-Y1158F expressing cells. These observations suggest that the level of IRS-1 phosphorylation rather than the level of IRS-2 phosphorylation mediates insulin-induced glycogen synthesis and DNA synthesis in 3T3-L1 pre-adipocytes.
Telting
D
D
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
van der Zon
G C
GC
Dorrestijn
J
J
Maassen
J A
JA
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D004247:DNA; D004815:Epidermal Growth Factor; D006003:Glycogen; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin; D011993:Recombinant Fusion Proteins; D013936:Thymidine; D014443:Tyrosine; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse