Insulin stimulates the phosphorylation of the 66- and 52-kilodalton Shc isoforms by distinct pathways.
In contrast to the 52-kDa Shc isoform, insulin stimulation caused a quantitative, time-dependent decrease in the SDS-PAGE mobility of 66-kDa Shc in both Chinese hamster ovary/IR cells and 3T3L1 adipocytes. Alkaline phosphatase treatment and direct phosphoamino acid analysis demonstrated that insulin stimulated an increase in serine phosphorylation of the 66-kDa isoform but not 52-kDa Shc, although the latter displayed a marked increase in tyrosine phosphorylation. To identify the responsible kinase pathway, we compared the effects on 66-kDa Shc serine phosphorylation by insulin, anisomycin, and osmotic shock, agents that specifically activate the ERK, JNK, or both pathways, respectively. Insulin and osmotic shock both stimulated a decrease in 66-kDa Shc mobility, whereas anisomycin had no effect. Furthermore, expression of a dominant-interfering Ras mutant (N17Ras) prevented the insulin-stimulated, but not the osmotic shock-induced serine phosphorylation of 66-kDa Shc. Consistent with a MEK-dependent pathway mediating 66-kDa Shc serine phosphorylation, the specific MEK inhibitor (PD98059) and expression of a dominant-interfering MEK mutant partially inhibited both the insulin and osmotic shock-induced reduction in 66-kDa Shc mobility. In contrast, expression of the MAP kinase phosphatase (MKP-1) completely prevented ERK activation but did not inhibit the serine phosphorylation of 66-kDa Shc. These data demonstrate that insulin stimulates the serine phosphorylation of the 66-kDa Shc isoform through a MEK-dependent mechanism.
Kao
A W
AW
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242, USA.
Waters
S B
SB
Okada
S
S
Pessin
J E
JE
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D008970:Molecular Weight; D009994:Osmolar Concentration; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007328:Insulin; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one