Cellular senescence is a tumor-suppressive process characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Passage of normal cells in culture leads to senescence, as do cellular stresses including ras oncoprotein expression and activation of the pRb tumor suppressor pathway. Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype in cultured cells, nor is it clear to what extent these processes occur in vivo. We have recently discovered a role for cdk5 in induction of SA-beta-gal expression and senescent cytoskeletal changes. Cdk5 activity increases in senescing cells and repression of the activity of the GTPase Rac1 by cdk5 is required for expression of SA-beta-gal. Cdk5 regulation of Pad activity is also necessary for actin polymerization accompanying senescent morphology in response to expression of pRb, activated ras, or continuous passage. Inhibition of cdk5 attenuates SA-beta-gal expression and blocks actin polymerization. We have further discovered that one substrate for cdk5 in senescent cells is the ERM protein ezrin. Expression of active pRb induces expression and altered localization of ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth. In order to more clearly understand the role of cdk5 and ERMs in senescence and tumor suppression, three specific aims are proposed: 1) Determine the mechanism of activation of cdk5 in senescent cells. 2) Determine how pRb activates transcription of the ezrin gene and use this information to understand transcriptional responses to pRb in senescent cells. 3) Ascertain the effects of constitutive cdk5 or ERM """"""""knock-down"""""""" or loss on proliferation and tumorigenesis. ? ? ? ?
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