Proteolytic cleavage of signal transduction molecules is an important mechanism for controlling cell growth, death and differentiation affecting a wide range of physiological and pathological processes. Intracellular proteolysis must be tightly regulated by endogenous inhibitors. Plasminogen activator inhibitor type 2 (PAl-2) is structurally and functionally a member of a large family of serine protease inhibitors or serpins. Serpins are key regulators of important biological processes such as complement activation, fibrinolysis, coagulation, cellular differentiation, tumor suppression, apoptosis and cell motility. PAl-2 was originally characterised as an inhibitor of the extracellular urokinase-type plasminogen activator, however PAl-2 is an inefficiently secreted serpin that exhibits a nucleocytoplasmic distribution. We have previously found that PAl-2 expression confers resistance to apoptosis and protects cells from certain cytopathic viruses in vitro. Our preliminary data identifies an intracellular activity for PAl-2 as a retinoblastoma tumor suppressor (pRb) binding protein that protects pRb from proteolytic degradation. The pRb family of proteins is ubiquitous regulators of transcription and plays a critical role in controlling cell proliferation. The central hypothesis of this application is that intracellular PAl-2 stabilizes pRb and p130, and in doing so, promotes pRb mediated activities associated with cell cycle arrest and promotion of differentiation, decreased sensitivity to E2F1 dependent apoptosis, transcriptional regulation and tumor suppression. The specific hypotheses to be tested are: 1) that PAl-2 binds pRb and the related pocket protein, p130, 2) that PAl-2 inhibits proteolytic cleavage of pRb, thereby enhancing pRb levels and pRb mediated activities, and 3) that PAl-2 promotes survival of keratinocytes and endothelial cells via pRb mediated stabilization. These hypotheses will be tested by 1) characterising the specific molecular interactions between PAl-2 and pRb utilizing mutant proteins in which specific functions have been disrupted, 2) determining the molecular mechanism by which PAl-2 stabilizes pRb and evaluating the role of calpain-like proteases in mediating proteolytic cleavage of pRb, and 3) testing the in vivo function of PAl-2 in stabilizing pRb using a PAl-2-/- mouse model. Analyses will specifically evaluate the roles of PAl-2 in keratinocyte differentiation, and endothelial cell proliferation and angiogenesis.
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