Ras inactivation of the retinoblastoma pathway by distinct mechanisms in NIH 3T3 fibroblast and RIE-1 epithelial cells.

Kevin Pruitt, Richard G. Pestell, and Channing J. Der

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Fig. 3.   Ras utilizes Raf-independent pathways to up-regulate cyclin-associated kinase activity in RIE-1 cells. A, exponentially growing cells stably expressing each DNA were harvested, and cyclin D1-associated kinase assays were performed. Cyclin D1 was immunoprecipitated, phosphorylation of recombinant GST-Rb fusion protein was measured by an in vitro kinase assay, and phosphorylation was quantitated using PhosphorImager analyses. B, Ras utilizes Raf-independent pathways to up-regulate cyclin E-associated kinase activity. Exponentially growing RIE-1 cells stably expressing the indicated protein were harvested, cyclin E-associated kinase activity was determined by immunoprecipitation (IP) of cyclin E, and phosphorylation of histone H1 was determined in an in vitro kinase assay. Recombinant Sf9 insect cell-expressed cyclin-E/CDK2 was assayed in parallel as an internal control for the in vitro kinase assay. Ras utilizes Raf-independent pathways to up-regulate cyclin E-associated kinase activity. C, Ras, but not Raf, activation causes an increase in CDK4 kinase activity. Exponentially growing RIE-1 cells stably expressing the indicated protein were harvested, CDK4 was immunoprecipitated, and phosphorylation of recombinant GST-Rb fusion protein was determined in an in vitro assay. Data shown are representative of at least three independent experiments.





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