Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

The Department of Cell BiologyRas Knockouts Aidin Defining OncogenicContribution of Normalc-RasAlan Wolfman, Ph.D.We are testing the hypothesis that thedifferent Ras isoforms (N-, Ha-, K(A),and K(B)-Ras) perform uniquefunctions. Using N and K(i)-Ras knockout celllines, we will test whether normal c-Ras isoformsare required for the transformation by specificoncogenic Ras proteins. Previous data from ourlaboratory support the hypothesis that transformationby oncogenic Ha-Ras, at protein levelsresembling those found in human tumors, requiresthe function of c-N-Ras. Oncogenic Ha-ras putsforth signals through an unidentified target tosecrete an EGF receptor ligand, which thenactivates c-N-Ras, Raf-1 and the MAP kinasecascade. This is the first report of Ras isoformcooperativity.Using cell lines established from N-Rasknockout mice, we have observed that c-N-Rasplays a pivotal role in regulating the cellularapoptotic set point. In the absence of c-N-Ras,mouse fibroblasts are hypersensitive to allapoptotic agents tested, including serumwithdrawal.In contrast, cells lacking c-K-Rasexpression are insensitive to the induction ofapoptosis. Together, these data suggest thatcellular Ras isoforms provide steady-state(“tonic”) signaling that regulates cells’ sensitivityto apoptotic agents. We are investigating themechanisms through which each of the cellularRas isoforms regulated the apoptotic mechanisms.In a second project, we are testing thehypothesis that c-N-Ras is distributed amongcellular organelles in discrete signaling modules.In quiescent cells we find all the plasma membraneassociated c-N-Ras complexed to Raf-1 andPKCe. We have also demonstrated that c-N-Rasis associated with membrane compartments otherthan the plasma membrane, such as the mitochondria.We are currently examining the role of c-N-Ras within each organelle and whether the c-N-Ras in specific organelles is associated with aunique Ras binding partner that might be requiredfor organelle-specific functions.Our laboratory is also investigating thesignaling systems that might be regulated bypulsed electromagnetic fields (PEMFs) that havea clinical outcome on patients having non-unionbone fractures. Using primary osteoblasts and anFDA-approved device that generates PEMFs, weare currently determining the initial biologicaloutcomes that are PEMF dependent. Using thisbiological outcome in cultured cells, we will thenexamine the signaling systems that are activatedupon PEMF treatment of primary osteoblasts.THE WOLFMANLABORATORYRESEARCH ASSOCIATESThomas PattersonSarah PlanchonPOSTDOCTORAL FELLOWSJinhui LiaoJanice WolfmanWolfman, J.C., and A. Wolfman (2000) Endogenous c-N-Ras provides a steady-state anti-apoptotic signal.J. Biol. Chem. 275:19315-19323.Hamilton, H. Liao, J., Cathcart, M., and A. Wolfman (2001) Constitutive association of c-N-Ras with Raf-1and protein kinase Ce in latent signaling modules. J. Biol. Chem. 276:29079-29090.Karasarides, M., Anande-Apte, B., and A. Wolfman (2001) A direct interaction between oncogenic Ha-Rasand phosphatidylinositol-3 kinase is not required for Ha-Ras-dependent transformation of epithelial cells. J.Biol. Chem. 276:39755-39764.Wolfman, J.C., Palmby, T., Der, C.J., and A. Wolfman (2002) Cellular N-Ras promotes cell survival bydownregulation of Jun N-terminal protein kinase and p38. Mol. Cell Biol. 22:1589-1606.83