Correct integration of signals coming from growth factors and from cell adhesion to the extracellular matrix (ECM) controls normal cellular proliferation, movement and differentiation, while aberrant integration contributes to many pathological states, including m a lignant neoplasia. Understanding the mechanism through which this integration occurs is crucial to our understanding the biology of transformed cells. The ability of growth factors to activate the MAPK cascade, a signaling pathway important for the regulation of cell proliferation and motility, is profoundly dependent on cell adhesion and the integrity of the cortical actin cytoskeleton, and is an archetype of adhesion-dependent signal transduction. It has recently been demonstrated that the anchorage-dependence of growth factor-mediated MAPK activation is coordinately regulated by the cAMP-dependent protein kinase (PKA), a venerable protein known to inhibit growth factor signaling as well as disrupt the actin cytoskeleton, and p21- activated kinase-1 (PAK), an important regulator of cortical actin and cell motility, through a mechanism that is likely to involve direct phosphorylation and inactivation of PAK by PKA. This proposal is designed to investigate further the newly described biochemical connection between PKA and PAK and its r o l e in the regulation of anchorage-dependent signal transduction. Experiments proposed in Aim 1 will define the mechanism of PAK inhibition by PKA-mediated phosphorylation and will examine the role of PKA in the PAK- dependent regulation of morphology and motility.
Aim 2 comprises experiments that will determine the mechanism through which PKA and PAK control anchorage- dependent signaling to MAPK as well as assess the ability of this signaling to promote downstream events leading to cell cycle entry and proliferation. Both a i m s w ill entail biochemical techniques including ionization mass spectrometry, co-immunoprecipitation and in vitro kinase and phosphatase assays, as well as cell biological techniques including immunofluorescence, migration assays, and soft agar colony formation. Evaluating the role of PKA and PAK in the integration of extracellular signals will broaden the current understanding of the complexity of anchorage-dependent growth and may provide new avenues for therapeutic intervention for malignant disease.
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