Protein kinase C epsilon-induced phosphoproteome

Protein kinase C (PKC) is a phospholipid-dependent serinehreonine kinase family, consisting of at least ten closely related isozymes. The different PKC isozymes play important roles in diverse signal transduction pathways. The exact role of each isozyme and their downstream targets are not known at present. Therefore, the elucidation of the roles of various PKC isozymes is important. The PKC epsilon (PKCeps) isozyme is widely expressed throughout the human body and seems to play pivotal roles in the function of the nervous, immune and cardiac systems. The highest expression levels of PKCeps have been found in the brain. PKCeps has been reported to be enriched at the growth cones of extending neurites in differentiating neural cells, to participate in nerve growth factor signaling as well as in transmitter release and to participate in cell death and survival. Since only very few substrates of PKCeps have been identified with reasonable certainty so far, the identification of PKC downstream targets represents the next major area of research in this field. It has been shown that in PC-12 pheochromocytoma cells, a widely used model system for neuronal studies, PKCeps is involved in the differentiation process to a neuronal phenotype, induced by nerve growth factor (NGF). In this application, investigations into differentiating PC-12 cells, to identify novel physiological substrates of PKCeps by quantitative phosphoproteomics and to verify the role as PKCeps substrates in the signaling cascade, are proposed. It is intended to elucidate the function of PKCeps in the NGF-induced differentiation of PC-12 cells, as a model system for the function of this isozyme in neurons. It is proposed to compare (1) proliferating PC-12 control cells with (2) cells in which differentiation is induced with NGF, with (3) cells treated with NGF and the PKCeps-inhibitory peptide EAVSLKPT (Johnson et al., 1996), and (4) ZFN-generated (Zinc Finger Nuclease), NGF induced, PKCeps knock-out cells. Optional, cells will be treated with the small molecule inhibitor PKCe141, an isozyme specific PKCeps inhibitor, which was developed in the lab of the applicant (patent application pending). State of the art techniques in quantitative phosphoproteomics will be employed to obtain the first high-resolution snapshot of the range of PKCeps phosphorylation events that occur in the cell immediately after NGF stimulation. Since PKCeps is an important sensitizing kinase in the CNS and an attractive drug target for treatment of several diseases such as cancer, inflammation, addiction, pain or anxiety disorders, the discovery of additional drug targets through identification of PKCeps substrates and the elucidation of signaling pathways involved, might help to find novel points of attack for therapeutic intervention and to manifest the importance of PKCeps in the nervous system.