Phosphorylation of proteins by specific protein kinases is one of the most common regulatory mechanisms that controls both the normal and aberrant behavior of cells. Oncogenes are frequently either protein kinases or part of a signal transduction pathway that regulates the activity of protein kinases. Thus, tracing the regulatory cascade that results in phosphorylation and establishing the function of phosphoproteins are two of the central focuses of oncology and developmental biology. This proposal seeks to identify the role of specific protein kinases in mediating the action of well defined regulators of nerve cell differentiation and growth. These studies use the PC12 cell line which is a cloned line derived from a transplantable pheochromocytoma. This line has proved to be a useful model system to study the regulation of neural differentiation. Many key differentiated properties including neurite formation, cell division, gene expression, neurotransmitter synthesis and neurotransmitter secretion are regulated in these cells by NGF, aFGF, bFGF, EGF, and adenosine. Adenosine probably acts by regulating the production of cAMP, but the role of the two distinct forms of the cAMP-dependent protein kinases are not yet understood. The involvement of specific protein kinases, including the cAKs, in the action of these peptide growth factors needs to be established. The primary objectives of this grant are to use somatic cell genetics to rigorously establish: 1) if unique roles can be ascribed to the two forms of the cAKs, 2) if all the actions of adenosine are mediated by the cAMP cascade, 3) if the cAKs play any essential role in the action of NGF, aFGF or bFGF. This approach, which we have already used effectively, requires us to isolate and characterize mutant cell lines that have defined deficits in the cAMP cascade; and to determine the effect of these mutations on the ability of the cell to respond to the peptide growth factors, adenosine, or defined pharmacological agents. We will also study an NGF-responsive kinase that we have discovered with the objectives of determining: 1) if it is casein kinase 2 or a closely related kinase, and 2) testing the hypothesis that it is responsible for one of the key NGF-dependent phosphorylation that occurs in vivo.
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