We are interested in understanding how normal intestinal cells regulate their growth and how loss of that regulation results in malignant transformation. Our research focuses on molecular mechanisms by which the Src tyrosine kinases and their inhibitors contribute to the regulation. We discovered two fundamental mechanisms whereby Src activity is regulated. One is by addition of a phosphate to a highly conserved tyrosine in the C-terminal tail. Mutation of this site converts the normal cellular Src into a transforming protein. The other is by interaction with Rack1, an endogenous substrate and inhibitor of Src kinases and colonic cell growth. Our recent in vitro studies demonstrate that Rack1 regulates colonic cell growth by suppressing Src activity at critical cell cycle checkpoints and during apoptosis. We hypothesize that Rack1 also regulates cell growth in vivo and plays a key role in mitochondrial apoptosis. To test these hypotheses we will:
Aim 1 : Analyze mechanisms by which Rack1 regulates growth of intestinal epithelia in vivo. A mouse model with Rack1 deleted in intestinal epithelia will be generated to do so. Rack1's influence on proliferation of crypt and stem cells, migration, differentiation and apoptosis of epithelial cells gene expression and development of intestinal neoplasia will be assessed. We will determine whether loss of Src rescues Rack1 deficiency in intestinal epithelia.
Aim 2 : Further analyze Rack1 function in mitochondrial cell death. Studies focus on mechanisms by which Rack1 engages and activates proapoptotic Bax and Bim, induces their translocation to mitochondria, mediates their interaction with each other and with prosurvival Bcl-2 family members, regulates Bax sequestration at mitochondria and facilitates Bax oligomerization. Endogenous inhibitors of oncogenic kinases that work at key checkpoints in the cell cycle, in proliferating crypt and stem cells and during apoptosis would exert powerful and pervasive control over cell growth. Exploitation of these multiple functions could be used to develop new and more powerful and selective strategies for treatment of human colon cancer.
of this proposal to public health lies in discovery of a fundamental mechanism by which the growth of intestinal cells is regulated;by a protein, Rack1, which inhibits a family of cancer-causing enzymes at key checkpoints in the cell cycle (before cells are fated to divide), in proliferating crypt cells and during programmed cell death. Such inhibitors yield powerful and pervasive control over cell growth;they are tumor suppressors and represent exciting new targets for colon cancer therapy.
|Mamidipudi, V; Cartwright, C A (2009) A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways. Oncogene :|
|Mamidipudi, Vidya; Miller, Laura D; Mochly-Rosen, Daria et al. (2007) Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells. Biochem Biophys Res Commun 352:423-30|
|Miller, Laura D; Lee, Kelly C; Mochly-Rosen, Daria et al. (2004) RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling. Oncogene 23:5682-6|
|Mamidipudi, Vidya; Chang, Betty Y; Harte, Rachel A et al. (2004) RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells. FEBS Lett 567:321-6|
|Chang, Betty Y; Cartwright, Christine A (2003) Detection of protein kinase-binding partners by the yeast two-hybrid analysis. Methods Mol Biol 233:327-43|
|Chang, Betty Y; Harte, Rachel A; Cartwright, Christine A (2002) RACK1: a novel substrate for the Src protein-tyrosine kinase. Oncogene 21:7619-29|
|Chang, B Y; Chiang, M; Cartwright, C A (2001) The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1. J Biol Chem 276:20346-56|
|Walter, A O; Peng, Z Y; Cartwright, C A (1999) The Shp-2 tyrosine phosphatase activates the Src tyrosine kinase by a non-enzymatic mechanism. Oncogene 18:1911-20|
|Chang, B Y; Conroy, K B; Machleder, E M et al. (1998) RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells. Mol Cell Biol 18:3245-56|