ROLE OF CYTOPLASMIC P27KIP1 IN CELL MOTILITY AND METASTASIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。转移性肿瘤生长是导致癌症患者死亡的主要原因，然而，其分子基础目前尚不清楚。转移性肿瘤细胞的迁移速度通常比正常细胞或非转移性肿瘤细胞快。这一观察结果导致了一个假设，即增加的细胞运动性（迁移）是细胞转移所必需的。最近，细胞周期蛋白CDK抑制剂p27KIP1在细胞运动中的一种新的细胞质依赖性作用被发现。结果表明，HGF信号通过Met受体导致p27 KIP1核输出到细胞质中。这种细胞质定位是p27 KIP1诱导细胞运动和导致肌动蛋白细胞骨架重排所必需的。这些结果表明，肿瘤细胞中信号的失调诱导了p27的细胞质输入。我们的中心假设是，在细胞质中，p27组装成一个复合物，参与细胞骨架重塑和细胞迁移，导致转移增加。胞质再定位和p27参与肌动蛋白细胞骨架重排在进化上与酵母中的α因子信息素信号传导相似。酵母细胞周期蛋白：cdk抑制剂Far1p介导细胞核内G1细胞周期阻滞和细胞质内shmoo形成，使肌动蛋白细胞骨架朝向相反的交配伴侣。作为对α因子的响应，Far1p从细胞核转移到细胞质，并与Cdc24p （Cdc42p的鸟嘌呤核苷酸交换因子）相互作用。与这种功能类比一致，p27散射域中存在的far1p样序列基序突变使细胞迁移和肌动蛋白重排失活，但保留了p27的细胞周期阻滞功能。我们的目的是通过tap标签纯化和质谱分析鉴定细胞质中与p27结合的蛋白复合物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Metastasic tumor growth is the principal event leading to death in patients with cancer, however, its molecular basis is presently poorly understood. Metastasic tumor cells generally migrate at a faster rate than normal cells or non-metastasic tumor cells. This observation leads to the hypothesis that increased cell motility (migration) is necessary for cells to become metastasic. Recently, a novel cytosolic-dependent role for the cyclin:CDK inhibitor p27KIP1 in cell motility was identified. It was shown that HGF signaling via the Met receptor resulted in the nuclear export of p27 KIP1 into the cytoplasm. This cytosolic localization was required for p27 KIP1 to induce cell motility and resulted in actin cytoskeletal rearrangements. These results suggest that deregulated signaling in tumor cells induce cytoplasmic import of p27. Our central hypothesis is that in the cytoplasm, p27 assembles into a complex involved in cytoskeletal remodeling and cell migration resulting in increased metastasis. The cytoplasmic relocalization and involvement of p27 in actin cytoskeletal rearrangement are evolutionarily reminiscent of alpha factor pheromone signaling in yeast. Far1p, the yeast cyclin:cdk inhibitor, mediates both a G1 cell cycle arrest in the nucleus and induction of shmoo formation in the cytoplasm that orients the actin cytoskeleton toward the opposite mating partner . In response to alpha factor, Far1p translocates from the nucleus to the cytoplasm and interacts with Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Consistent with this functional analogy, mutation of a Far1p-like sequence motif present in the p27 scatter domain inactivates both cell migration and actin rearrangement, but preserves the cell cycle arrest functions of p27. Our objective is to identify the proteins complexes binding to p27 in the cytoplasm by TAP-tag purification and mass spectrometry analysis.