RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints

Abstract

Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G 1 and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G 1 and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.

A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways

“…Consistent with these results, depleting Src also induces apoptosis in nearly a third of cells. Previously, we showed that overexpression of wild type but not mutant RACK1 inhibits Src kinase activity in HT-29 cells (Mamidipudi et al, 2007). Collectively, these results show that RACK1, in part through its inhibitory influence on Src, induces apoptosis of HT-29 cells.…”

“…Using a yeast two-hybrid assay, we identified RACK1 as a novel substrate and binding partner of Src and an endogenous inhibitor of Src kinase activity and cell growth (Chang et al, 1998(Chang et al, , 2001(Chang et al, , 2002. We showed that RACK1 exerts its effect on colonic cell growth, in part by suppressing Src activity at G 1 and mitotic checkpoints (Mamidipudi et al, 2004a(Mamidipudi et al, , 2007. We hypothesize that RACK1 regulates other aspects of cell growth.…”

“…Earlier we showed that RACK1 regulates colonic cell growth, in part by suppressing Src activity at G 1 and mitotic checkpoints (Mamidipudi et al, 2004a(Mamidipudi et al, , 2007. Another potential mechanism by which RACK1 could regulate growth is by inducing apoptosis.…”

“…As a result, apoptotic cells have a lower DNA content and staining with propidium iodide will show these cells in the sub-G 0 G 1 region. To determine whether potential RACK1 effects are dependent on RACK1's ability to inhibit Src activity, we used an RACK1 mutant (RACK1 Y228F/ Y246F) that lacks the Src phosphorylation and binding sites and consequently is no longer a substrate, binding partner, or an inhibitor of Src (Chang et al, 2002;Mamidipudi et al, 2004aMamidipudi et al, , 2007. We observed that 18% of cells overexpressing wild-type RACK1 were in the sub-G 0 G 1 region (Figure 1a).…”

Cellular Localization, Invasion, and Turnover Are Differently Influenced by Healthy and Tumor-Derived Extracellular Matrix

“…51 Decreased proliferation of seeded cells was observed at later time points, indicating the influence of the ECM on the cell cycle. Indeed, in our system the CRC-derived ECM induces high proliferation of transformed epithelial cells, and the healthy-derived ECM show a constraining effect on cell proliferation, indicating that the different ECMs induced different selective pressures on transformed cells.…”