Insulin stimulates the phosphorylation of the 66- and 52-kilodalton Shc isoforms by distinct pathways

Endocrinology. 1997 Jun;138(6):2474-80. doi: 10.1210/endo.138.6.5203.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adaptor Proteins, Signal Transducing*
  • Adaptor Proteins, Vesicular Transport*
  • Adipocytes / metabolism
  • Animals
  • CHO Cells
  • Cricetinae
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Flavonoids / pharmacology
  • Insulin / pharmacology*
  • Kinetics
  • MAP Kinase Kinase Kinase 1*
  • Mice
  • Molecular Weight
  • Osmolar Concentration
  • Phosphorylation
  • Phosphoserine / metabolism
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / isolation & purification
  • Proteins / metabolism*
  • Shc Signaling Adaptor Proteins
  • Signal Transduction*
  • Src Homology 2 Domain-Containing, Transforming Protein 1


  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Enzyme Inhibitors
  • Flavonoids
  • Insulin
  • Proteins
  • Shc Signaling Adaptor Proteins
  • Shc1 protein, mouse
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Phosphoserine
  • Protein-Serine-Threonine Kinases
  • MAP Kinase Kinase Kinase 1
  • Map3k1 protein, mouse
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one