PKCalpha-Mediated Mechanisms in Metastatic Melanoma

概述：蛋白激酶c - α (PKCa)激活促转移信号通路，其直接底物及其下游成分定义不清。pkca介导途径的一些生物学终点包括：细胞运动的获得、细胞周期活性的改变、蛋白酶的释放和氧化还原平衡的扰乱。由于细胞中存在多种PKC异构体以及数百种其他蛋白激酶，在可追踪激酶方法出现之前，确定特定蛋白激酶的底物一直很困难。本应用程序的目的是使用可追踪激酶方法鉴定PKCa的直接蛋白底物，并澄清由每个磷酸化蛋白控制的表型结果。此外，介导细胞粘附和运动的PKCa底物将被进一步研究作为抑制黑色素瘤转移潜能的相关靶点。将进行突变底物（显性阴性或伪磷酸化）的工程表达，以建立由蛋白质底物磷酸化引起的生物学终点（Specific Aim 1）。除了运动和粘附、增殖和细胞周期活性以及细胞骨架结构的表型分析外，该项目还将利用扫描电化学显微镜（SECM）根据还原氧化反应性区分高转移和弱转移黑色素瘤细胞。氧化还原反应性是一种与PKCa活性相关的表型，它提供了一种新技术，可用于评估PKCa底物对细胞氧化还原状态的影响。与新底物并行，表型分析将使用已知的PKCa底物，MARCKS（豆芽糖酰化丙氨酸-富c激酶底物）蛋白（Specific Aim 2）进行。用PKCa底物的功能增加或丧失突变体稳定转染的黑色素瘤细胞将通过体内实验转移测定来评估转移潜力的变化。磷酸化蛋白的免疫化学测试将在具有不同转移潜力的小鼠和人类黑色素瘤细胞以及已知临床病理的人类黑色素瘤标本中进行（Specific Aim 3）。这项研究将产生机制和实用信息，可用于实施更精细的化疗策略和转移性黑色素瘤的诊断筛选。该应用程序将研究蛋白激酶c - α (PKCa)的蛋白质底物，PKCa是一种酶，其活性对黑色素瘤细胞在全身扩散（“转移”）的能力至关重要。“可追踪激酶方法”将用于鉴定PKCa的直接蛋白底物，并表征这些磷酸化蛋白介导的生物学终点。重点将放在与黑色素瘤细胞转移潜力相关的端点上。这项工作将揭示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黑色素瘤。

DESCRIPTION (provided by investigator): Proposal Summary: Protein kinase C-alpha (PKCa) activates pro-metastatic signaling pathways of which the immediate substrates and their downstream components are poorly defined. Some biological endpoints of PKCa-mediated pathways include: the acquisition of cell movement, alteration of cell cycle activity, release of proteinases, and perturbation of the redox balance. Because there exist multiple PKC isoforms, as well as hundreds of other protein kinases in the cell, determination of substrates for a specific protein kinase had been difficult until the development of the Traceable Kinase Method. The objective of this application is to identify direct protein substrates of PKCa using the Traceable Kinase Method, and to clarify the phenotypic outcomes governed by each phospho-protein. Furthermore, PKCa substrates that mediate cell adhesion and motility will be further investigated as relevant targets through which to suppress the metastatic potential of melanoma. Engineered expression of mutant substrates (dominant-negative or pseudo-phosphorylated) will be performed to establish biological endpoint(s) that result(s) from the phosphorylation of a protein substrate (Specific Aim 1). In addition to phenotypic assays of motility and adhesion, proliferation and cell cycle activity, and cytoskeletal structure, this project will harness scanning electrochemical microscopy (SECM) to distinguish between highly metastatic and weakly metastatic melanoma cells on the basis of their reduction-oxidation reactivity. The redox reactivity is a phenotype that is linked to PKCa activity, and offers a novel technology that can be used to assess the impact of a PKCa substrate on the cellular redox status. In parallel with new substrates, phenotypic analysis will be carried out with a known PKCa substrate, the MARCKS (myristoylated alanine-rich C-kinase substrate) protein (Specific Aim 2). Melanoma cells that have been stably transfected with a gain- or loss-of-function mutant of a PKCa substrate will be assessed for a change in metastatic potential by in vivo experimental metastasis assay. Immunochemical testing of a phospho-protein will be performed with murine and human melanoma cells of differing metastatic potential, and with human melanoma specimens of known clinical pathology (Specific Aim 3). This research will yield both mechanistic and practical information that can be used to implement a more refined chemotherapeutic strategy and diagnostic screening for metastatic melanoma. This application will investigate the protein substrates of protein kinase C-alpha (PKCa), an enzyme whose activity is critical to the ability of melanoma cells to spread throughout the body ("metastasis"). The "Traceable Kinase Method" will be used to identify direct protein substrates of PKCa, and the biological endpoints mediated by these phosphoproteins will be characterized. An emphasis will be placed on those endpoints related to the metastatic potential of melanoma cells. This work will reveal new chemotherapeutic targets and disease-related biomarkers for control and detection of metastatic human melanoma.