Our effort to identify candidate genes responsible for the nonproliferating senescent phenotype led us to characterize a 57 kDa nonproliferation-specific nuclear protein, statin, which is phosphorylated by an associated 45 kDa serine/threonine kinase (p45), and forms a complex with this enzyme and another unphosphorylated protein of 110 kDa (p110), identified so far as the retinoblastoma gene product, RB. We hypothesize that in nongrowing cells p57 statin may function as a sequester to prevent the phosphorylation of the associated p110 RB; because the lack of p34(cdc2) kinase alone does not block RB phosphorylation in early G1, we suggest that this p45 kinase may be the missing enzyme needed for this step. During early G1 phase, rapid statin loss releases p45 kinase to phosphorylate RB; in nongrowing cells such as senescent fibroblasts, permanent statin presence sequesters p45, and along with the absence of p34(cdc2) is part of the mechanism producing a complete blockade of RB phosphorylation, with cascade effects culminating in inability of DNA synthesis. We suggest that a """"""""two-hit RB antiphosphorylation"""""""" model may pertain in nongrowing cells; this proposal aims to investigate how statin functions to block RB phosphorylation, and whether molecular manipulations might remove this blockade in nonreplicating cells, or implement it in their replicating counterparts.
Specific aims i nclude: (1.) investigating the specific cellular action regulating the binding reaction of p57 statin, p45 statin kinase, and p110 RB in nongrowing cells; (2.) purifying p57 and p45 from senescent cells, for protein microsequencing and functional studies; (3.) molecular cloning, sequencing and characterization of p57; (4.) investigating the molecular regulation of p57 expression during in vitro aging; (5.) early functional analysis of p57 and p45 in RB phosphorylation in early G1 phase; and (6.) early functional analysis of the presence of p57 and absence of cdc2 kinase in RB phosphorylation in senescent cells. Our hypothesis that statin may function as the sequester of the kinase needed for early RB phosphorylation provides the first handle to begin the investigation of kinases involved in early G1 phase, and offers an intriguing and workable model in the continuing research quest to understand the molecular mechanisms controlling the cessation of proliferation in in vitro aged human fibroblasts. Answers to this ques- tion will add to our knowledge of cell cycle control, a fundamental mechanism pivotal to the wellbeing of significant processes such as terminal differentiation and cellular aging.
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