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. ? ? ?
