Functional importance of Shc tyrosine 317 on insulin signaling in Rat1 fibroblasts expressing insulin receptors.
Shc is phosphorylated on Tyr-317, which serves as a docking site for Grb2. To investigate the specific role of Shc phosphorylation and Shc.Grb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substitution (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). From different clonal cell lines, cells expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compared with endogenous Shc were chosen for analysis of insulin signaling. Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in WT-Shc cells. Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compared with that in HIRc cells. Likewise, reduction of endogenous Shc expression by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation. Although 317Y/F-Shc was also able to interact with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicating that 317Y/F-Shc functions as a dominant-negative mutant. The kinetics of mitogen-activated protein (MAP) kinase activation closely paralleled the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Shc cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells. Importantly, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine and bromodeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphorylation plays an important role, via coupling with Grb2 and competition with IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.
Ishihara
H
H
First Department of Medicine, Toyama Medical & Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan.
Sasaoka
T
T
Ishiki
M
M
Takata
Y
Y
Imamura
T
T
Usui
I
I
Langlois
W J
WJ
Sawa
T
T
Kobayashi
M
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D007328:Insulin / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D008938:Mitosis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013329:Structure-Activity Relationship; D014443:Tyrosine; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D011506:Proteins; D012333:RNA, Messenger; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases