Identifying endosomal Phosphatidylinositol 4-phosphate 5-kinase isoforms regulating growth factor stimulated integrin recycling and migration

项目总结 通常情况下，上皮细胞是不活动的，但可以在生长因子信号作用下发生迁移。 生长因子刺激的迁移在发育中起重要作用，是修复的早期步骤 在组织损伤后进行处理。然而，在癌症进展过程中，生长因子信号可以成为 通过致癌突变或肿瘤微环境中基质细胞的作用而异常激活。 生长因子刺激癌细胞的迁移促进转移和进一步的疾病进展。一个 彻底了解启动迁移以响应生长因子信号的信号是至关重要的 开发干预措施以促进伤口愈合期间的迁移或抑制粘连和 癌细胞的迁移。焦点粘连将细胞附着到细胞外基质上，并允许产生 细胞用来迁移的牵引力。必然地，增长因素刺激的移民需要增加 由整合素组织的新的焦点粘连的组装。生长因子刺激导致 将先前内化的整合素快速循环至质膜。之前在Santy实验室的工作 证明了细胞粘素活性是刺激整合素循环所必需的，而且只有那些细胞粘素 与磷脂酰肌醇4，5-二磷酸(PI(4，5)P2)结合的剪接变异体可以启动这种转运过程。 PI(4，5)P2在需要的地方通过磷脂酰肌醇4-磷酸5-激酶从PI4P局部产生 (PIP5Ks)，暗示PIP5Ks参与了整合素的刺激循环。以前关于这些方面的工作还没有得到充分的研究 激酶主要集中在它们在质膜上的作用，在信号转导和 膜蛋白的内化。拟议的研究将确定需要哪些PIP5K亚型 用于刺激整合素循环，并确定它们在内体系统中的哪个位置产生PI(4，5)P2以 启动受刺激的整合素循环。将使用击倒和救援策略来测试PIP5K的影响 整合素回收中的剪接异构体。这些异构体的作用位置将由定位决定。 两种异构体及其产物的PI(4，5)P2。在正交法中，化学诱导的二聚化 将用于招募生产和消耗PI(4，5)P2的酶到各个内体隔室，以 确定在什么地方需要局部生产这种脂质，以刺激整合素返回细胞表面。 了解导致生长因子刺激的整合素循环的步骤将有助于开发 在不影响生长因子信号整体的情况下调节这一过程的干预。

PROJECT SUMMARY Epithelial cells are normally non-motile but can become migratory in response to growth factor signaling. Growth factor stimulated migration plays important roles in development and is an early step in the repair process after tissue damage. During cancer progression however, growth factor signaling can become aberrantly activated through oncogenic mutation or the actions of stromal cells in the tumor microenvironment. Growth factor stimulated migration of carcinoma cells promotes metastasis and further disease progression. A thorough understanding of the signals that initiate migration in response to growth factor signaling is essential for developing interventions to either enhance migration during wound healing or inhibit adhesion and migration of cancer cells. Focal adhesions attach cells to the extracellular matrix and allow the generation of traction forces that cells use to migrate. By necessity, growth factor stimulated migration requires increased assembly of new focal adhesions, which are organized by integrins. Growth factor stimulation leads to the rapid recycling of previously internalized integrins to the plasma membrane. Previous work in the Santy lab demonstrated that cytohesin activity is required for stimulated integrin recycling and that only those cytohesin splice variants that bind phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) can initiate this trafficking process. PI(4,5)P2 is produced locally where needed from PI4P by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), implicating PIP5Ks in the stimulated recycling of integrins. Previous work on these understudied kinases has predominately focused on their actions at the plasma membrane during signaling and internalization of membrane proteins. The proposed studies will determine which PIP5K isoforms are required for stimulated integrin recycling and determine where within the endosomal system they produce PI(4,5)P2 to initiate stimulated integrin recycling. A knockdown and rescue strategy will be used to test the impact of PIP5K splice isoforms on integrin recycling. The location where these isoforms act will be determined by localization of both the isoforms and their product, PI(4,5)P2. In an orthogonal approach, chemically induced dimerization will be used to recruit PI(4,5)P2 producing and consuming enzymes to individual endosomal compartments to determine where local production of this lipid is required to stimulate the return of integrins to the cell surface. Understanding the steps that lead to growth factor stimulated integrin recycling will allow development of interventions to modulate this process without affecting growth factor signaling as a whole.