Characterization of membrane-associated substrates of Ca2+-dependent kinases in astrocytes

Membrane-associated kinase substrates are likely transducers of extracellular signals elicited by neuroimmunomodulators and other signaling molecules. Whereas specific signal transduction pathways in astrocytes are being defined, the global view is lacking. We, therefore, characterized membrane-associated substrates of Ca2+-dependent kinases in primary astrocytes using 2-dimensional gel electrophoresis. Ten proteins were phosphorylated in vitro and characterized with respect to their relative molecular mass (in the range 10 kDa - 100 kDa), isoelectric point (range 4.2 - 9.0) and four conditions of phosphorylation. They varied broadly in their requirements for phosphorylation displaying distinct kinase preferences. Eight phosphoproteins were substrates of protease kinase C. Judging by abundance and intensity of phosphorylation, the principal PKC substrates were three acidic proteins associated with the plasma membrane. These results suggest that a relatively small number of membrane-associated proteins serve as transducers of signals mediated by Ca2+-dependent kinases and most of them are PKC substrates in astrocytes.