Shc isoform-specific tyrosine phosphorylation by the insulin and epidermal growth factor receptors.
Insulin stimulation of Chinese hamster ovary cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IR/ER) resulted in the tyrosine phosphorylation of the 52-kDa Shc isoform with a relatively low extent of 46-kDa Shc tyrosine phosphorylation. In contrast, EGF stimulation resulted in the tyrosine phosphorylation of both the 52- and 46-kDa Shc isoforms. Consistent with these differences, Grb2 predominantly bound to the 52-kDa Shc isoform following insulin stimulation, whereas Grb2 associated with both the 52- and 46-kDa Shc isoforms after EGF stimulation. Further, in vitro kinetic analysis demonstrated that the insulin receptor has a 4-fold greater Vmax with no significant difference in the Km for the purified 52-kDa Shc isoform compared with the 46-kDa Shc isoform. However, the EGF receptor displayed the identical Vmax and Km for tyrosine phosphorylation of both of these species. In direct contrast to the EGF receptor, we also observed significant differences in binding interactions between the insulin receptor with the 52- and 46-kDa Shc isoforms in vitro. These data demonstrate that the predominant insulin-dependent Shc signaling pathway occurs via the 52-kDa Shc isoform, whereas the EGF receptor can effectively use both the 52- and 46-kDa Shc species.
Okada
S
S
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242, USA.
Yamauchi
K
K
Pessin
J E
JE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives*
D017931:DNA Primers; D007328:Insulin; D007527:Isoenzymes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011972:Receptor, Insulin