RACK1 Regulates G1/S Progression by Suppressing Src Kinase Activity

When regulated, the Src tyrosine kinase participates in a wide array of signaling pathways that control cell proliferation, differentiation, adhesion and survival (reviewed in references 2, 18, 39, and 51). Deregulated Src is oncogenic (10, 32, 40, 41). Thus, identifying mechanisms that regulate Src will reveal important information about how normal cells regulate their growth. RACK1 is the founding member of a family of receptors for activated C kinase (PKC) collectively called RACKs (reviewed in references 22 and 34). Following serum or platelet-derived growth factor (PDGF) stimulation or PKC activation, we observed that RACK1 colocalizes with Src at the plasma membrane and functions as a substrate, binding partner, and inhibitor of Src (as measured in vitro) and as a growth inhibitor in NIH 3T3 cells (11-13). RACK1 inhibits cell growth, in part, by prolonging the G0/G1 phase of the cell cycle. G1/S transition is controlled by two key families of proteins: cyclin-dependent kinases (CDKs) and cyclins. Cyclins bind to and activate CDKs (21, 46). Another critical event in cell cycle progression at G1/S is phosphorylation and inactivation of the retinoblastoma protein (pRb) and release of regulatory proteins such as E2F, which stimulate transcription of target genes that are required for cell proliferation. v-Src, the transforming homolog of c-Src, directly affects cell cycle proteins that regulate G1/S transition (26, 42, 51). For example, v-Src induces rapid transit through the G1 checkpoint and entry into S phase by simultaneously suppressing the CDK inhibitor p27 and inducing p21 and cyclins D1, E, and A (42, 49). Growth factors trigger cascades of intracellular signals that lead to induction of the nuclear oncogene Myc (3, 23, 36). Cyclin/CDK complexes are then activated, and cells pass the G1 checkpoint and embark on DNA replication. c-Src activation is essential for PDGF-induced G1/S transition and DNA replication (4, 5, 44, 51, 53). Kinase-inactive Src induces a G1/S block that can be rescued by constitutive expression of Myc (4). Moreover, microinjection of antibodies that inhibit Src family kinases or treatment with a selective Src family kinase inhibitor, SU6656, inhibits PDGF- and Src-driven Myc induction and DNA synthesis (5, 44). Collectively, these results show that Src activity regulates the transcriptional activation of Myc and G1/S transition. We hypothesized that RACK1 prolongs the G0/G1 phase of the cell cycle by inhibiting the activity of Src, and thereby Myc and other cell cycle regulators, at the G1 checkpoint. We found that RACK1 overexpression induces a partial G1 arrest by suppressing Src activity at the G1 checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and CDK4 and CDK2 are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G1/S progression is delayed. Conversely, short interference RNA (siRNA) inhibition of RACK1 expression activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G1/S transition. RACK1 suppresses Src- but not mitogen-activated protein kinase (MAPK)-dependent PDGF signaling.