Protein kinase C substrates that drive motility of cancer cells

As the intracellular receptor of tumor promoting phorbol esters, protein kinase C (PKC) is functionally linked to carcinogenesis and metastasis. Therefore, it is crucial to identify substrates of PKC in order to understand the mechanisms by which these substrate proteins participate in cancer-related phenotypes such as motile behavior. The work to be described consists of two projects: (1) new PKC substrates that contribute to the motility phenotype of human breast cells, and (2) the role of a known PKC substrate, MARCKS (Myristoylated Alanine- Rich C-Kinase Substrate) in the motility pathway of mouse melanoma cells.;To identify direct substrates, a chemical-genetic approach was used to engineer the ATP binding site of PKC-alpha, delta and &zgr; to enable them to bind an unnatural ATP analogue. Consequently, phosphorylation is attributed exclusively to the mutant enzyme, and the phosphorylated protein bands are potential substrates. Following expression in human breast cells (MCF-10A), co-immunoprecipitation of the mutant enzyme bound with high affinity protein substrates was carried out. Following addition of an ATP analogue, a number of phosphorylated proteins were produced and subsequently analyzed by mass spectrometry. PKC-alpha and delta traceable kinases had very similar phosphorylation patterns, whereas the profile for PKC-&zgr; was distinct. Several potential protein substrates involved in cytoskeletal structure were identified for PKC-alpha and PKC-delta, including proteins that bind small GTP-binding proteins (Rho Kinase-1, Cdc42ep4, Rho/cdc42/Rac activating protein-1), as well as proteins that had been previously documented as PKC substrates (IQGAP, VASP).;In a second project, the role of MARCKS was investigated in the context of mouse melanoma cell motility. As a protein that is known to crosslink actin bundles to the plasma membrane, this PKC substrate has been linked with cell migration and adhesion. Upon phosphorylation by PKC, MARCKS is released into the cytoplasm, thereby promoting actin rearrangement. Weakly metastatic B16 F1 cells do not express detectable phospho-MARCKS. However, F1 cells that are engineered to express a pseudo-phosphorylated mutant of MARCKS exhibit elevated motility. When F1 cells are treated with okadaic acid (OA), an inhibitor of protein phosphatases, increases in both phospho-MARCKS and motility are observed. OA-induced motility can be substantially eliminated when cells are pre-treated with a shRNA reagent to knock down MARCKS expression, or when expressing a phosphorylation-resistant mutant of MARCKS. Furthermore, a phosphorylation-resistant MARCKS mutant that was made exclusively cytoplasmic due to lack of a myristoyl group was observed to inhibit motility of OA-treated F1 cells as well as motility of highly metastatic melanoma cells (mouse F10 and human A375 cells). These findings imply that MARCKS promotes motility through cytoplasmic interactions involving its phosphorylated effector domain. It is concluded that phospho-MARCKS has a previously undocumented role in the cytoplasm that promotes motile behavior and possibly the metastatic potential of many different cancer cells.;Overall, this work describes substrates of PKC that transmit the motility signal in cancer cells, and suggests novel targets for the development of anti-metastasis agents.