The plasma membrane Ca2+-ATPases (PMCA) play a primary role in cellular regulation owing to their ability to remove Ca2+ with high affinity. PMCAs are composed of a large family of closely related isoforms derived by differential splicing of primary transcripts of at least four distinct genes in mammals. The structural differences thus far identified between isoforms occur only in regions involved in regulation of activity by calmodulin, phospholipids and protein kinases. Their expression is tightly regulated in intact animal cells both at the level of the gene and RNA processing. That artificial inhibition of PMCA1 expression by antisense vector transfection inhibits the ability of PC-6 cells to produce normal neuritic processes in response to NGF was recently shown. Loss of PMCA1 is accompanied by loss of alpha1-integrin expression and concomitant loss of adherence properties as well as substantial decrease in ionomycin-mediated calcium fluxes and increase in glucocorticoid (dexamethasone) dependent reporter gene expression. Polypeptides migrating with the same relative molecular weight as PMCAs on SDS-PAGE are heavily tyrosine phosphorylated in wt and sense transfected PC-6 cells, but are absent in the antisense transfectants. Tyrosine phosphorylation of PMCA1/4 has been shown both in in vitro studies with purified components and in human platelets in response to physiological stimulation. These results strongly that the plasma membrane Ca2+-ATPase is regulated by tyrosine phosphorylation in some settings, and that it may play a direct role in signal transduction involving tyrosine kinases. A combination of biochemical, immunological, pharmacological and recombinant DNA approaches will be used to elucidate the nature, generality and exact function of this apparent tyrosine phosphorylation in regulating plasma membrane calcium pump activity both in vitro and in vivo. Studies with stably transfected Rat1 cells expressing pp60src species, as well as, wt and defective focal adhesion kinase will examine the potential role of this pathway in mediating regulation of PMCA through tyrosine phosphorylation. Yeast two hybrid selection procedures will be used to identify additional PMCA directed tyrosine kinases. The resulting tyrosine kinase(s) and corresponding purified PMCA isoforms, prepared by a combination of classical and recombinant DNA approaches, will be used in detailed analyses of PMCA regulation through tyrosine phosphorylation and its role in cell signaling pathways.
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