The long-term goals of this proposal are to define the mechanism of regulation of the 70kD-S6 protein kinases (pp70S6k) in response to a variety of growth modulators including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), insulin, interleukin2 (IL-2) and tumor promoting phorbol esters. These growth modulators have been previously shown to regulate pp70S6k activity,, but how this occurs and what role the signalling pathway that regulates pp70S6k plays in the physiological response of cells to these factors in not known. Our immediate goals are to generate and express by various means, different forms of pp70S6k (wild type, constitutively-activated and dominant-interfering) in cultured cells to help define the roll of this protein kinase in cell physiological processes; to further define how pp7OS6k is regulated by phosphorylation; to identify and characterize the pp70S6k-protein kinases; and to identify and characterize other components of this important signalling system. Defining the components that regulate pp70S6k activity and the further characterization of the enzyme itself may lead not only to a better general understanding of cell growth control and its obvious relevance to the cancer problem, but also may be particularly relevant with regards to our understanding of the immune system and insulin responsiveness. Recent genetic studies in Drosophila indicate that the 40S ribosomal protein S6 (the only known target of pp70S6k) may act as a tumor suppressor in the immune system. This observation may be related to the inability of unphosphorylated S6, within the 40S subunit, to translate specific mRNA with unique 5' untranslated regions. The hypothesis is that S6 phosphorylation or removal of the protein would relieve this inhibition of translation. Additionally, the ability of the T cell immunosuppressant rapamycin to potently inhibit IL-2-dependent T cell proliferation and very specifically, pp70S6k activity, suggests an important role for pp70S6k in T cell proliferation. One of the apparent defects in some insulin-resistant humans is the inability of a normal insulin receptor to activate pp70S6k as seen in insulin-responsive humans. Additionally, specific inhibitors of the growth-regulated lipid kinase phosphatidylinositol 3-kinase, not only antagonize IL-2-, insulin-, PDGF- and NGF-mediated activation of pp70S6k, but also interfere with insulin- regulated translocation of the glucose transporter GLUT4, PDGF-stimulated actin cytoskeleton reorganization and IgE-mediated histamine release in different cell systems. Thus it is clear that defining this signalling pathway will improve our understanding of how pp70S6k is regulated and its potential downstream effects on translation, transcription and in general growth control, as well as improve our understanding of processes regulating protein sorting/secretion and cell shape.
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