Abstract
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 βIIPKC, thereby affecting βIIPKC translocation and function. We found that the association of βIIPKC 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 βIIPKC. 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.
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Acknowledgements
We thank Toufan Parman for critical review of the manuscript. This work was supported by grants from the National Institutes of Health to CAC (R01 DK43743) and DM-R (R01 HL52141). LDM was supported, in part, by NIH Digestive Disease Center grant DK65339.
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Miller, L., Lee, K., Mochly-Rosen, D. et al. RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling. Oncogene 23, 5682–5686 (2004). https://doi.org/10.1038/sj.onc.1207735
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DOI: https://doi.org/10.1038/sj.onc.1207735