REGULATION OF SEQUENTIAL PROTEIN KINASE PATHWAYS

National Jewish Health, Denver CO

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Abstract

PC12 pheochromocytoma cells undergo a reversible growth arrest and differentiation program to a sympathetic neuron-like phenotype in response to the signal transduction process initiated by specific growth factor receptors. Primary responses to growth factor receptor activation resulting in the onset of PC12 cell neuronal differentiation involves the phosphorylation of proteins on both tyrosine and serine-threonine residues. Thus, the regulation of the phosphorylation cascades controlling the progression of neuronal differentiation determines PC12 cell phenotype. The stimulation and control of the phosphorylation and second messenger cascades that induce PC12 cell differentiation are genetically manipulated by expression of mutant genes for receptor kinases (PDGF receptor), protein phosphatases (phosphotyrosine and serine/threonine) and G proteins (Go and Gs). Using this approach, the influence of transfected dominant activating or dominant negative mutant gene products on the biochemical pathways controlling PC12 cell phenotype is systematically analyzed. Because a network of protein kinases and second messengers are activated during growth factor stimulated PC12 cell differentiation, particular emphasis is placed on the control of kinase cascades by protein phosphatases. Altered protein phosphatase expression and activity allows the selective manipulation of arms of the protein kinase network in order to define their involvement in control of cell phenotype. A great deal of research has focused on the role of specific protein kinases in growth and differentiation control, and the ability to define and manipulate protein phosphatase activities provides an opportunity to investigate the consequence of altered dephosphorylation control on cell function. Work in this proposal focuses on the control of biochemical pathways involved in a neuronal differentiation program, but understanding the biochemical mechanisms which control hormone and growth factor receptor signal transduction pathways is necessary to define the molecular basis for many diseases. The reversible nature of the PC12 cell differentiation program regulated by growth factor receptors, which induce mitogenic responses in most cell types, provides the unique ability to investigate the biochemical relationships between growth and differentiation.