RACK1, A RECEPTOR FOR ACTIVATED C-KINASE AND A HOMOLOG OF THE BETA-SUBUNIT OF G-PROTEINS, INHIBITS ACTIVITY OF SRC TYROSINE KINASES AND GROWTH OF NIH 3T3 CELLS

To isolate and characterize proteins that interact with the unique dom ain and SH3 and SH2 domains of Src and potentially regulate Src activi ty, we used the yeast two-hybrid assay to screen a human lung fibrobla st cDNA library. We identified RACK1, a receptor for activated C kinas e and a homolog of the beta subunit of G proteins, as a Src-binding pr otein. Using GST-Src fusion proteins, we determined that RACK1 binds t o the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was de monstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vit ro kinase activity of Src in a concentration-dependent manner. GST-RAC K1 (2 mu M) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serin e/threonine kinases that we tested, Tyrosine phosphorylation on many c ellular proteins decreased in 293T cells that transiently overexpresse d RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyse s revealed that RACK1 overexpressing cells do not show an increased ra te of necrosis or apoptosis but do spend significantly more time in G( 0)/G(1) than do wild-type cells. Prolongation of G(0)/G(1) could accou nt for the increased doubling time of RACK1 overexpressing cells. We s uggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.