REGULATION OF RB PROTEIN FUNCTION BY CYCLIN-DEPENDENT

视网膜母细胞瘤是一种由突变引起的发育中的视网膜的恶性肿瘤 一个基因的两个等位基因（RB1）。 RB1是典型的肿瘤 抑制基因，一种其失活导致肿瘤发生的基因。 不 只有RB1的突变会导致视网膜母细胞瘤，但它的突变已经被证实。 在多种常见的人类肿瘤中检测到。 其中包括癌症 乳腺、肺、前列腺、膀胱和子宫颈。 由于这些原因， 已经成为人们努力了解分子机制的焦点， 调节细胞向恶性转化 RB1函数，如果可以的话 适当地操纵，可以作为一种手段， 癌的 这种方法的先决条件是了解RB1如何 监管. RB1基因产物（110RB）是一种核磷蛋白， 通过细胞分裂周期的G1期调节进展。 110 RB的磷酸化与细胞周期同步发生， 提示110RB功能可能受细胞周期依赖性调节， 磷酸化 细胞周期蛋白依赖性激酶（cdks）是很好的候选者 对于调节110RB的激酶，由于110RB的特征振荡， 它们在细胞周期中的活动。 之前，我们已经证明， 显微注射未磷酸化的110RB或截短形式（p56RB）， 早期G1细胞将阻止进入S期。 利用这种分析，我们建议 直接评估G1和S相磷酸化的后果 细胞周期蛋白/cdk复合物对RB1蛋白负调控能力的影响 S阶段进入。 本提案中提出的假设是， 特异性细胞周期蛋白依赖性激酶磷酸化抑制110 RB 功能 该提案的具体目标是：1）产生 纯化的110RB的制备物，其已经在体外被 细胞周期蛋白依赖性激酶; 2）测试这些差异的能力， 磷酸化110RB制剂以抑制细胞周期进程; 3） 确定重要的特定磷酸化靶位点， cdks的调节; 4）表征突变的功能后果 5）测试这些CDK调节细胞的能力 RB1样蛋白p107的周期阻滞活性。 初步结果表明，在体外110 RB的磷酸化， 细胞周期蛋白D1/cdk4（D1/k4）、细胞周期蛋白E/cdk2（E/k2）和细胞周期蛋白A/cdk2（A/k2） 在SDS-PAGE解析时引起特征迁移率变化， 表示过度磷酸化。 D1/k4引起的110 RB过度磷酸化 抑制其阻止细胞周期的能力。 RB1蛋白 被A/k2或E/k2过度磷酸化的蛋白保留了其阻断进入 s期 经这些激酶处理的110 RB的胰蛋白酶磷酸肽图谱 揭示了一种复杂的磷酸化模式 虽然模式产生了 D1/k4磷酸化与E/k2磷酸化部分重叠 和A/k2、D1/k4磷酸化位点，导致四个胰蛋白酶 磷酸肽仅被E/k2和A/k2弱磷酸化。 本提案的长期目标是了解如何 磷酸化调节RB1功能，激酶能够 调节RB1，以及这与其他RB1样调节的关系 proteins.

Retinoblastoma is a malignancy of the developing retina caused by mutation of both alleles of a single gene (RB1). RB1 is the prototypical tumor suppressor gene, a gene whose inactivation leads to tumorigenesis. Not only does mutation of RB1 cause retinoblastoma, but its mutation has been detected in a variety of common human neoplasia. These include carcinoma of the breast, lung, prostate, bladder, and cervix. For these reasons it has become the focus of efforts to understand the molecular mechanisms that regulate a cell's transition to malignancy. RB1 function, if it could be appropriately manipulated, might serve as a means to treat relevant cancers. A prerequisite for this approach is understanding how RB1 regulated. The RB1 gene product (110RB) is a nuclear phosphoprotein which negatively regulates progression through G1 phase of the cell division cycle. Phosphorylation of 110RB occurs in synchrony with the cell cycle, suggesting that 110RB function may be regulated by cell cycle-dependent phosphorylation. The cyclin-dependent kinases (cdks) are good candidates for kinases that regulate 110RB due to the characteristic oscillation of their activity during the cell cycle. Previously, we have shown that microinjection of unphosphorylated 110RB, or a truncated form (p56RB), in early G1 cells will block entry into S phase. Using this assay, we propose to directly assess the consequences of phosphorylaiton by G1 and S phase cyclin/cdk complexes on the ability of RB1 protein to negatively regulate S phase entry. The hypothesis addressed in this proposal is that phosphorylation by specific cyclin dependent kinases inhibits 110RB function. The specific aims of this proposal are to; 1) produce preparations of purified 110RB that have been phosphorylated in vitro by cyclin-dependent kinases; 2) test the ability of these differentially phosphorylated 110RB preparations to inhibit cell cycle progression; 3) determine the particular phosphorylation target sites important for regulation by cdks; 4) characterize the functional consequences of mutation at these sites; 5) test the ability of these cdks to regulate the cell cycle arrest activity of RB1-like protein p107. Preliminary results indicate that in vitro phosphorylation of 110RB by cyclin D1/cdk4 (D1/k4), cyclin E/cdk2 (E/k2), and cyclin A/cdk2 (A/k2) causes the characteristic mobility shift upon resolution by SDS-PAGE, denoting hyperphosphorylation. Hyperphosphorylation of 110RB by D1/k4 inhibits its ability to arrest the cell cycle. However, RB1 protein hyperphosphorylated by A/k2 or E/k2 retains its ability to block entry into S phase. Tryptic phosphopeptide mapping of 110RB treated by these kinases revealed a complex pattern of phosphorylation. While the pattern produced by phosphorylation with D1/k4 partially overlaps that produced with E/k2 and A/k2, D1/k4 phosphorylates sites which lead to four tryptic phosphopeptides only weakly phosphorylated by E/k2 and A/k2. The long term objectives of this proposal are to understand how phosphorylation regulates RB1 function, which kinases are capable of regulating RB1, and how this relates to regulation of other RB1-like proteins.