Protein Kinase C Substrates in Human Breast Cancer

描述（由申请人提供）：蛋白激酶C（PKC）是EGF刺激（erbB 2）途径的上游组分，其活性与培养的人乳腺细胞的运动行为和转移行为的获得有关。 因此，PKC底物蛋白的鉴定将为开发抗转移化疗提供潜在的靶点。 最近，该实验室成功地将可追踪激酶方法应用于人乳腺细胞，确定α-微管蛋白是PKCa的底物，从而促进运动。 通过体外和细胞内方法测试的假设是α-微管蛋白在Ser-165处的磷酸化通过刺激微管伸长来促进运动性。 此外，该实验室已经发现MARCKS蛋白（先前被其他人定义为PKC底物）驱动人类乳腺细胞的运动行为，并依赖于磷酸-α-微管蛋白和微管进行细胞内运输。 计划的研究包括：具体目的（1）研究α-微管蛋白磷酸化对体外聚合、培养的人乳腺细胞中微管延伸和3D培养中形成的集落形态的意义。 具体目标（2）将探索MARCKS与细胞中微管的相互作用，这些细胞表达这两种PKC底物的突变构建体的荧光融合蛋白。 这些研究将采用运动分析和活细胞成像（共聚焦显微镜）。 对于特定目的（3），将应用可追溯激酶方法鉴别和比较其他PKC亚型（d和？）的底物在人乳腺细胞和肿瘤细胞中，以区分共享底物和那些亚型特异性底物。 这项工作将确定新的化疗靶点和疾病相关的生物标志物，用于控制和检测转移性人类乳腺癌。 公共卫生相关性：该提案将研究蛋白激酶C（PKC）的蛋白底物，PKC是一种酶，其活性对乳腺细胞在全身扩散（“转移”）的能力至关重要。 “可追踪激酶方法”将用于鉴定PKC的直接蛋白底物，并表征这些蛋白介导的生物学效应。 这项工作将确定新的化疗靶点和疾病相关的生物标志物，用于控制和检测转移性人类乳腺癌。

DESCRIPTION (provided by applicant): Protein kinase C (PKC) is an upstream component of the EGF-stimulated (erbB2) pathway whose activity has been linked to motile behavior of cultured human breast cells and to the acquisition of metastatic behavior. Therefore, identification of PKC substrate proteins would offer potential targets for developing anti-metastasis chemotherapies. Recently, this laboratory successfully applied the Traceable Kinase Method to human breast cells by determining that a- tubulin is a substrate of PKCa that consequently promotes motility. A hypothesis to be tested by both in vitro and intracellular approaches is that phosphorylation of a-tubulin at Ser-165 promotes motility by stimulating microtubule elongation. In addition, the MARCKS protein (previously defined by others to be a PKC substrate) has been found by this laboratory to drive motile behavior of human breast cells, and to depend on phospho-a-tubulin and microtubules for its intracellular trafficking. Planned studies are: Specific Aim (1) to investigate the significance of a-tubulin phosphorylation to polymerization in vitro, microtubule elongation in cultured human breast cells, and morphology of colonies formed in 3D cultures. Specific Aim (2) will explore the reciprocal interactions of MARCKS with microtubules in cells that express fluorescent fusion proteins of mutant constructs of these two PKC substrates. These studies will employ motility assays and live cell imaging (confocal microscopy). For Specific Aim (3), the Traceable Kinase Method will be applied to identify and compare substrates of additional PKC isoforms (d and ?) in human breast cells and tumor cells in order to distinguish shared substrates from those that are isoform-specific. This work will identify new chemotherapeutic targets and disease-related biomarkers for control and detection of metastatic human breast cancer. PUBLIC HEALTH RELEVANCE: This proposal will investigate the protein substrates of protein kinase C (PKC), an enzyme whose activity is critical to the ability of breast cells to spread throughout the body ("metastasis"). The "Traceable Kinase Method" will be used to identify direct protein substrates of PKC, and the biological effects mediated by these proteins will be characterized. This work will identify new chemotherapeutic targets and disease-related biomarkers for control and detection of metastatic human breast cancer.