Abstract
Changes in CrkII and CrkL phosphorylation are associated with insulin-like growth factor receptor activation in cultured cells. We examined whether similar changes also occur following administration of recombinant human insulin-like growth factor-I to the intact animal. In female rats starved overnight, CrkL phosphorylation was significantly increased 12 min after insulin-like growth factor-I administration. Tyrosine phosphorylation of CrkII was not detectable in either control or treated animals. Paxillin, a 65-70-kDa phosphoprotein containing high affinity binding sites common for the Src homology 2 (SH2) domains of CrkII and CrkL, was observed in both CrkII and CrkL immunoprecipitates. Insulin-like growth factor-I treatment stimulated the association of CrkII with paxillin. In contrast, the same treatment resulted in the dissociation of the CrkL-paxillin complex. Similar effects of insulin-like growth factor-I treatment on the association of CrkL with tyrosine phosphorylated paxillin were observed in fibroblasts overexpressing CrkL. This study demonstrates that the activation of the insulin-like growth factor-I receptor induces changes in the tyrosine phosphorylation and protein-protein interactions of the Crk proteins in vivo. The different responses of CrkL and CrkII to insulin-like growth factor-I receptor activation suggest distinct roles for these two adapter proteins in signal transduction.
Highlights
From the ‡Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1770 and the Section on Molecular Carcinogenesis, Department of Pathology, Childrens Hospital, Los Angeles, California 90027
insulin-like growth factors (IGFs)-I Increases insulin receptor substrate (IRS)-1 Tyrosine Phosphorylation—Prior to determining whether IGF-IR activation induced changes in Crk phosphorylation, we examined as a positive control the effect of IGF-I treatment on the tyrosine phosphorylation of IRS-1
Immunoblotting with an anti-IRS-1 antibody confirmed that approximately equal amounts of IRS-1 protein were being precipitated, with the phosphorylation data shown in Fig. 1B adjusted to account for any differences in the amount of IRS-1 protein in the precipitates
Summary
Changes in CrkII and CrkL phosphorylation are associated with insulin-like growth factor receptor activation in cultured cells. Phosphorylated IRS-1 acts as a docking protein for multiple signaling proteins including Grb, the p85 subunit of phosphoinositol-3Ј kinase, the phosphatase PTP1D (Syp), Nck, and CrkII [4, 5] Adapter proteins such as Grb, Nck, and Crk function to mediate protein-protein interactions and are believed to have an important role in regulating the activity of intracellular second messenger systems [6]. The guanine nucleotide exchange protein C3G is bound constitutively to the SH3 domains in CrkII and is believed to activate JNK, which has been shown to be essential for the oncogenic functions of v-Crk [9] Both Crk proteins have been shown to mediate the association of C3G with tyrosine phosphorylated Cbl [10, 11]. The development of the uterus is severely impaired in mice homozygous for a deletion of the IGF-I gene [19], while estrogen treatment of ovariectomized rats stimulates the tyrosine phosphorylation of the IGF-IR -subunit and IRS-1 [20]
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