Abstract
Insulin and epidermal growth factor (EGF) stimulation of Chinese hamster ovary cells expressing the human insulin and EGF receptors resulted in a time-dependent decrease in the ability of a Rap1 antibody (amino acid epitope 121-136) to immunoprecipitate Rap1 from whole cell detergent extracts. This was due to an apparent masking of Rap1 as heat denaturation of the whole cell detergent extracts (5 min at 100 degrees C) resulted in equal immunoprecipitation of Rap1 with this epitope-specific antibody. The time-dependent change in Rap1 immunoreactivity was paralleled with an insulin-stimulated dissociation of the CrkII-C3G complex. Similarly, EGF treatment also resulted in a time-dependent dissociation of the CrkII-C3G complex that occurred concomitant with the masking of the 121-136 Rap1 epitope. Furthermore, pretreatment of the cells with the tyrosine kinase inhibitor, genistein, decreased both the basal and insulin-stimulated tyrosine phosphorylation of CrkII that directly correlated with the amount of CrkII that was immunoprecipitated with C3G. Together, these data suggest that insulin and EGF stimulation result in the dissociation of the CrkII-C3G complex, thereby inducing an apparent conformation change in Rap1.
Highlights
Ras is a low molecular weight (Mr 21,000) GTP-binding protein that plays an essential role in cell proliferation and differentiation (1, 2)
Opposing the Ras activation pathway, the low molecular GTP-binding protein Rap was originally observed to revert or suppress the transformed phenotype in Ki-Ras-transformed fibroblasts (18, 19). This apparent antagonism between Ras and Rap function may reflect the ability of Rap and Ras to interact with the same downstream effectors, since these proteins share identical sequences within their respective effector domains (18 –20)
We demonstrate that insulin and epidermal growth factor (EGF) stimulation result in a rapid dissociation of the CrkII-C3G complex that correlates with an apparent conformational change and/or masking of Rap[1] immunoreactivity
Summary
Ras is a low molecular weight (Mr 21,000) GTP-binding protein that plays an essential role in cell proliferation and differentiation (1, 2). We demonstrate that insulin and EGF stimulation result in a rapid dissociation of the CrkII-C3G complex that correlates with an apparent conformational change and/or masking of Rap[1] immunoreactivity. Insulin stimulation had no significant effect on the total amount of Rap[1] protein that was detergent-solubilized in the whole cell extracts as detected by immunoblotting with a Rap1specific monoclonal antibody (Fig. 1, lanes 1–5).
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