Regulation and function of cdk5 and ezrin in senescence

描述(申请人提供)：细胞衰老是一种肿瘤抑制过程，其特征是不可逆的细胞周期退出、独特的形态和衰老相关的β-半乳糖苷酶(SA-β-GAL)的表达。正常细胞在培养中的传代会导致衰老，细胞应激也会导致衰老，包括ras癌蛋白的表达和pRb肿瘤抑制通路的激活。尽管细胞衰老具有潜在的生物学重要性，但人们对导致培养细胞衰老表型的机制知之甚少，也不清楚这些过程在体内发生的程度。我们最近发现了CDK5在诱导SA-β-Gal表达和衰老细胞骨架改变中的作用。在衰老细胞中，CDK5的活性增加，并且SA-β-Gal的表达需要CDK5抑制GTPase rac1的活性。CDK5对Pad活性的调节也是肌动蛋白聚合所必需的，同时伴随着pRb表达、激活的ras或连续传代的衰老形态。抑制CDK5可减弱SA-β-GAL的表达，并阻断肌动蛋白聚合。我们进一步发现，衰老细胞中CDK5的一个底物是ERM蛋白Ezrin。活性pRb的表达诱导Ezrin的表达和定位改变，Ezrin是一种参与膜-细胞骨架信号转导的肌动蛋白结合蛋白。PRb的表达刺激了CDK5介导的Ezrin的磷酸化，导致随后的膜结合和诱导细胞形状的改变，将pRb的活性与衰老细胞的细胞骨架调节联系起来。这些结果开始阐明衰老诱导和衰老形状变化的潜在机制，并描述了可能有助于衰老细胞影响肿瘤生长的新途径。为了更清楚地了解CDK5和ERMS在衰老和肿瘤抑制中的作用，提出了三个特定的目标：1)确定CDK5在衰老细胞中的激活机制。2)确定pRb如何激活Ezrin基因的转录，并利用这一信息来了解衰老细胞对pRb的转录反应。3)确定结构性CDK5或ERM的“敲除”或缺失对细胞增殖和肿瘤发生的影响。

DESCRIPTION (provided by applicant): 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.