GROWTH CONTROL IN AGING FIBROBLASTS

我们为确定候选基因所做的努力 非增殖性衰老表型使我们描述了一个57 kDa的 非增殖特异性核蛋白，他汀类，它是 被相关的45 kDa丝氨酸/苏氨酸激酶(P45)磷酸化，以及 与这种酶和另一种未磷酸化的蛋白质形成复合体 110 kDa(P110)，目前已鉴定为视网膜母细胞瘤基因产物Rb。 我们假设，在不生长的细胞中，p57他汀类药物可能作为一种 隔离剂，以防止相关的p110RB的磷酸化； 因为p34(Cdc2)蛋白的缺乏本身并不能阻断Rb G1期早期的磷酸化，我们认为这个P45激酶可能是 缺少这一步所需的酶。在G1期早期，快速抑制 在不生长的细胞中，Loss释放P45激酶来磷酸化Rb； 衰老的成纤维细胞，永久性的他汀类存在隔离物P45，和 伴随着p34(Cdc2)的缺失是产生 完全阻断Rb磷酸化，连锁效应达到顶峰 不能合成DNA。我们建议“两支安打的RB” “抗磷酸化”模式可能适用于不生长的细胞；这一建议 目的是研究他汀类药物如何作用于阻断Rb的磷酸化，以及 分子操作是否可以消除这一障碍 非复制型细胞，或在复制型细胞中实现它。 具体目标包括：(1)研究特定的细胞行为 调节p57他汀类蛋白、p45他汀类蛋白激酶的结合反应 不生长细胞中的p110RB；从衰老中提纯P57和P45 细胞，用于蛋白质微测序和功能研究；分子 P57基因的克隆、测序及鉴定；调查 体外衰老过程中p57表达的分子调控；早些时候 P57和p45在G1期早期Rb磷酸化中的功能分析 阶段；及(6.)P57和p57存在的早期功能分析 衰老细胞Rb磷酸化过程中cdc2蛋白的缺失。我们的 他汀类药物可能起到隔离所需激酶的作用的假说 对于早期Rb磷酸化提供了第一个句柄开始 参与早期G1期的激酶的研究，并提供了一种 耐人寻味且可行的模型在持续的研究探索中 了解控制心绞痛停止的分子机制 体外老化人成纤维细胞的增殖。这个问题的答案是- TION将增加我们对细胞周期控制的知识，这是一个基本的 对重大进程的福祉至关重要的机制，如 终末分化和细胞衰老。

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 include: (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.