title: Two naturally occurring insulin receptor tyrosine kinase domain mutants provide evidence that phosphoinositide 3-kinase activation alone is not sufficient for the mediation of insulin's metabolic and mitogenic effects.
abstract: We have recently reported (1) that two naturally occurring mutants of the insulin receptor tyrosine kinase domain, Arg-1174 --> Gln and Pro-1178 --> Leu (Gln-1174 and Leu1178, respectively), both found in patients with inherited severe insulin resistance, markedly impaired receptor tyrosine autophosphorylation, with both mutant receptors being unable to mediate the stimulation of glycogen synthesis or mitogenesis by insulin when expressed in Chinese hamster ovary cells. However, these mutations did not fully prevent IRS-1 phosphorylation in response to insulin in these cells, suggesting that IRS-1 alone may not be sufficient to mediate insulin's metabolic and mitogenic effects. In the present study, we have demonstrated that these mutations also impair the ability of the insulin receptor to activate the transcription factor Elk-1 and promote GLUT4 translocation to the plasma membrane. Although at low concentrations of insulin, the mutant receptors were impaired in their ability to stimulate the tyrosine phosphorylation of IRS-1, at higher insulin concentrations we confirmed that the cells expressing the mutant receptors showed significantly increased tyrosine phosphorylation of IRS-1 compared with parental nontransfected cells. In addition, at comparable insulin concentrations, the association of the p85alpha subunit of phosphoinositide 3-kinase (PI3-kinase) with IRS-1 and the enzymatic activity of IRS-1-associated PI3-kinase were significantly enhanced in cells expressing the mutant receptors. In contrast, no significant stimulation of the tyrosine phosphorylation of Shc, GTP loading of Ras, or mitogen-activated protein kinase phosphorylation was seen in cell lines expressing these mutant receptors. Thus, no activation of any measurable mitogenic or metabolic response was detectable, despite significant insulin-induced phosphorylation of IRS-1 and its association with PI3-kinase in cells stably expressing the mutant insulin receptors. These findings suggest that PI3-kinase activation alone may be insufficient to mediate a wide range of the metabolic and mitogenic effects of insulin. Additionally, the data provide support for the notion that insulin activation of Ras is more closely linked with Shc, and not IRS-1, phosphorylation.
authors.affiliation: Department of Medicine, Cambridge University, Addenbrookes Hospital, Hills Road, CB2 2QR, Cambridge, United Kingdom. sorahill@hgmp.mrc.ac.uk
authors.forename: A
authors.initials: A
authors.lastname: Krook
authors.forename: J P
authors.initials: JP
authors.lastname: Whitehead
authors.forename: S P
authors.initials: SP
authors.lastname: Dobson
authors.forename: M R
authors.initials: MR
authors.lastname: Griffiths
authors.forename: M
authors.initials: M
authors.lastname: Ouwens
authors.forename: C
authors.initials: C
authors.lastname: Baker
authors.forename: A C
authors.initials: AC
authors.lastname: Hayward
authors.forename: S K
authors.initials: SK
authors.lastname: Sen
authors.forename: J A
authors.initials: JA
authors.lastname: Maassen
authors.forename: K
authors.initials: K
authors.lastname: Siddle
authors.forename: J M
authors.initials: JM
authors.lastname: Tavaré
authors.forename: S
authors.initials: S
authors.lastname: O'Rahilly
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D005260:Female; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D007930:Leucine / Q000737:chemistry; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry* / Q000235:genetics; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013329:Structure-Activity Relationship
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D005973:Glutamine; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D007930:Leucine