CHARACTERIZATION OF PROTEIN-KINASE AND PHOSPHATASE SYSTEMS IN CHICK CILIARY GANGLION

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary gang lion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2/phospholipid-dependent protein kinase activities were detected in hom ogenates of ciliary ganglion dissected from 15-16-day-old embryos. Aut ophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dep endent protein kinase II in the presence of Ca2+/calmodulin or 5 mM Zn SO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electro phoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose mo lecular weights corresponded to the regulatory subunits of cyclic AMP- dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following in cubation with [gamma-P-32]ATP under most conditions had an apparent mo lecular weight of 65,000 which corresponds to the chicken form of myri stoylated alanine-rich C kinase substrate, a known substrate of protei n kinase C. Another substrate for protein kinase C was a 45,000 molecu lar weight protein which was tentatively identified as neuromodulin (B -50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP- dependent protein kinase were detected, protein kinase A strongly labe lled a 40,000 molecular weight protein. Using P-32(i)-labelled glycoge n phosphorylase, protein phosphatases 1 and 2A were identified in cili ary ganglia homogenates at levels which were indistinguishable from fo rebrain at the same age. The major endogenous protein substrates in ci liary ganglion homogenates from 15-16-day-old embryos were also labell ed to a similar extent in homogenates of ciliary ganglia from newly ha tched chickens. Intact ciliary ganglia remained viable for several hou rs after dissection and, after incubation with P-32(i), responded to p horbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterizati on of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms r esponsible for regulating synaptic transmission in this tissue.