RACK1-mediated novel signaling pathways in growth and apoptosis

To elucidate novel RACK1-mediated signaling pathways, we performed yeast two-hybrid screen to identify novel interacting partners of RACK1. IGF-1R and DLC1 were found to interact with RACK1 in yeast.; In Chapter 3, we demonstrate a novel role of RACK1 as an adaptor for insulin and insulin-like growth factor 1 receptor (IGF-1R)-mediated STAT3 activation specifically. Intracellular association of RACK1 via its N-terminal WD domains 1 to 4 (WD1-4) with insulin receptor (IR)/IGF-1R is augmented upon respective ligand stimulation, whereas association with STAT3 is constitutive. Purified RACK1 or RACK1 WD1-4 associates directly with purified IR, IGF-1R, and STAT3 in vitro. Insulin induces multiprotein complex formation of RACK1, IR, and STAT3. Overexpression or downregulation of RACK1 greatly enhances or decreases, respectively, IR/IGF-1R-mediated activation of STAT3 and its target gene expression. Site-specific mutants of IR and IGF-1R impaired in RACK1 binding are ineffective in mediating recruitment and activation of STAT3 as well as in insulin- or IGF-1-induced protection of cells from anoikis. RACK1-mediated STAT3 activation is important for insulin and IGF-1-induced anchorage-independent growth in certain ovarian cancer cells. We conclude that RACK1 mediates recruitment of STAT3 to IR and IGF-1R specifically for activation, suggesting a general paradigm for the need of an adaptor in mediating activation of STATs by receptor protein tyrosine kinases.; In Chapter 4, we found that RACK1 formed a complex with DLC1 and BimEL in the presence of apoptotic agents, including paclitaxel and stausporine. RACK1 inhibited the release of BimEL from microtubule and also mediated the degradation of BimEL upon paclitaxel treatment, most likely through ElonginB/C-Cullin2-CIS E3 ligase complex at the centrosome. Both RACK1 and CIS were important for proteasome-mediated BimEL degradation and overexpression of CIS resulted in a dose dependent reduction of BimEL. CIS and BimEL level displayed a negative correlation in most of the ovarian and breast cancer lines and tumor specimens. RACK1 mediated paclitaxel resistance of breast cancer cells in vitro and in vivo. Our study suggests a role of RACK1 in protecting cancer cells from apoptosis by regulating the release and degradation of BimEL, which may lead to drug resistance in chemotherapy.