RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling.
RACK1 is the founding member of a family of receptors for activated C kinase collectively called RACKs. Upon activation of PKC, RACK1 co-localizes with the Src tyrosine kinase at the plasma membrane and functions as a substrate, binding partner and inhibitor of Src (as measured in vitro), and a growth inhibitor in NIH 3T3 cells. To further analyze the function of RACK1 in Src and PKC signaling, we utilized cell-permeable peptides that modulate the interaction of RACK1 and betaIIPKC, thereby affecting betaIIPKC translocation and function. We found that the association of betaIIPKC and RACK1 is necessary for Src phosphorylation of RACK1. Src activity is required for tyrosine phosphorylation of RACK1, and for RACK1 binding to Src, but not to betaIIPKC. Endogenous Src kinase activity, as measured by phosphorylation of Sam68 (a mitotic-specific Src substrate involved in cell cycle regulation and RNA splicing) or p190RhoGAP (a Src substrate and GTPase-activating protein involved in actin reorganization), increases with disruption of the Src-RACK1 complex, and decreases with enhanced complex formation. RACK1 inhibits Src-mediated p190RhoGAP signaling and actin cytoskeleton rearrangement. Thus, RACK1 functions as an endogenous inhibitor of the Src kinase in diverse signaling pathways that regulate distinct cellular functions. Our results demonstrate the potential for using peptide modulators of Src activity as a tool for uncovering the function of Src in cells.
Miller
Laura D
LD
Department of Medicine, Stanford University, Stanford, CA 94305, USA.
Lee
Kelly C
KC
Mochly-Rosen
Daria
D
Cartwright
Christine A
CA
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins; D011493:Protein Kinase C / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C513041:Arhgap35 protein, mouse; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C513253:Khdrbs1 protein, mouse; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D012097:Repressor Proteins; D019061:src-Family Kinases; D011493:Protein Kinase C