The research proposed here opens a new line of investigation in studies aimed to elucidate molecular mechanisms that regulate differentiation, maturation and intercellular interactions in developing neurons of the embryonic central nervous system. A large body of evidence documents the significant role of protein phosphorylation activity in the regulation of neuronal development and adaptation. These studies have delineated events that occur subsequent to the activation of receptors by neurohormones and growth factors, and focus on the activity of intracellular protein kinases. Of potential equal importance are findings demonstrating that neurons possess an ecto-protein kinase activity which phosphorylates extracellular proteins localized at the outer surface of the plasma membrane. Recent studies have implicated this activity in processes underlying neuritogenesis, but a systematic investigation on the role of ecto-protein kinase in neuronal development has not been carried-out yet. The research proposed here will fill this gap. We have identified two specific protein substrates (M.W. of 11.7K and 13K) of neuronal ecto-protein kinase whose extracellular phosphorylation is uniquely enhanced during the early phase of development of embryonic brain neurons. The Research Plan of the present proposal outlines experiments designed to obtain the data-basis, biochemical and molecular tools and probes essential for direct determination of the function of these specific extracellular protein phosphorylation systems. Accordingly, the specific aims of this proposal are: (1) Purification to apparent homogeneity of the neuronal ecto-protein kinase from embryonic brain. (2) Isolation of the 11.7K and 13K phosphoprotein substrates of this ectokinase, whose phosphorylation by extracellular ATP is unique to early developmental phases. (3) Preparation of polyclonal and monoclonal antibodies that will inhibit selectively the extracellular phosphorylation of the 11.7K and 13K proteins at the surface of developing neurons. (4) Cloning and sequencing of the complementary DNA for these 11.7K and 13K surface phosphoproteins. The purified kinase and the functional antibodies will enable us to determine conclusively the role of these specific extracellular protein phosphorylation in neuritogenesis measured in embryonic neurogenesis in the brain in-vivo. It is anticipated that this project will provide significant information on a novel regulatory mechanism operating in differentiating neurons, that may be a site of molecular alteration in certain pathophysiological conditions underlying abnormal central nervous system development.
