Analysis of protein kinase activities in rabbit ciliary processes: Identification and characterization using exogenous substrates

Abstract

Protein-kinase activities in rabbit ciliary process tissue were characterized and quantitated using histone, casein, and myosin light chain as substrates. At least four different protein-kinase activities were separated and identified in the supernatant (soluble) and in the particulate fraction using DEAE-cellulose ion-exchange chromatography. Typical activities of the protein kinases in ciliary processes dissected from one eye were as follows: in the supernatant fraction; protein kinase C, 185.0 pmol min-1; cyclic AMP-dependent protein kinase type II, 34.0 pmol min-1; casein kinase type II, 85.1 pmol min-1; protein kinase M, 9.8 pmol min-1: in the particulate fraction; protein kinase C, 55.1 pmol min-1; cyclic AMP-dependent protein kinase type II, 12.5 pmol min-1; casein kinase type II, 13.4 pmol min-1, and protein kinase M, 5.5 pmol min-1. No cyclic GMP-dependent and no calmodulin-dependent protein-kinase activities were detectable using histone, casein or myosin light chain as substrates. The apparent molecular weight of protein kinase C as estimated by exclusion chromatography on a column of Sephadex G-200 was about 90,000. Inhibitory and stimulatory effects of recently synthesized isoquinolinesulfonamide derivatives (H-7 and H-8), heparin, and polylysine were studied in ciliary process protein kinases. H-7 and H-8 were potent inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and protein kinase M, (IC50 less than 10 microM) but had no inhibitory effects on casein kinase. Heparin at 4 micrograms ml-1 inhibited casein kinase activity almost completely without affecting cyclic AMP-dependent or protein kinase C activities. Poly D- or L-lysine were both found to activate (approximately double) casein kinase activity at 40 micrograms ml-1, but did not significantly activate cyclic AMP-dependent protein kinase or protein kinase C. These results provide basic information on the protein kinase enzymes in the ciliary process and show that protein kinase C is the major kinase in this tissue. This suggests a possible role of the Ca2+ and protein kinase C system in transport functions of ciliary processes and in the regulatory mechanism of aqueous-humor formation additional to the already established importance of the cyclic AMP-dependent protein-kinase enzyme.