title: 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.
abstract: To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 microM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells. Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.
authors.affiliation: Department of Medicine, Stanford University, Stanford, California 94305, USA.
authors.forename: B Y
authors.initials: BY
authors.lastname: Chang
authors.forename: K B
authors.initials: KB
authors.lastname: Conroy
authors.forename: E M
authors.initials: EM
authors.lastname: Machleder
authors.forename: C A
authors.initials: CA
authors.lastname: Cartwright
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D004789:Enzyme Activation; D016193:G1 Phase; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D016192:Resting Phase, Cell Cycle; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D010455:Peptides; D000075370:Receptors for Activated C Kinase; C070301:peptide I; D014443:Tyrosine; D019061:src-Family Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins