Abstract
The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.
Highlights
From the Diabetes Branch, NIDDK Laboratory of Cellular and Molecular Biology, and the §NCI, National Institutes of Health, Bethesda, Maryland 20892
The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells
insulin receptor substrate-1 (IRS-1) and 4PS function as multisite “docking proteins” which link insulin and IGF-I receptors as well as IL-4 receptors [9, 21, 25, 26] with various post-receptor signaling systems
Summary
From the Diabetes Branch, NIDDK Laboratory of Cellular and Molecular Biology, and the §NCI, National Institutes of Health, Bethesda, Maryland 20892. Crk proteins share homology with the adaptor proteins Grb and Nck [2, 3], and are known to associate with two guanine nucleotide releasing proteins, C3G and mSos [4, 5] These studies suggested that Crk may participate in growth factor-induced activation of Ras. In addition, we have recently shown that stimulation of IGF-I1 receptors results in rapid tyrosine phosphorylation of Crk [6]. Given that stimulation with IGF-I results in tyrosine phosphorylation of Crk [6] and the interaction of IRS-1 with the Crk-related molecules Grb and Nck, it was of interest to determine whether IRS-1 and 4PS associate with Crk. In the present study we find that c-Crk associates with IRS-1 and 4PS and enhances IGF-I-induced mitogenesis
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