Casein kinases and their protein substrates in rat liver cytosol: evidence for their participation in multimolecular systems.

We have shown by gel filtration on Sepharose 4B at low ionic strength that casein kinases S (type 1), heparin-insensitive, and TS (type 2), heparin-inhibited, of rat liver cytosol participate in two distinct multimolecular systems, Ve/Vo = 1.25 and Ve/Vo = 1.90, respectively, both less retarded than the peak of cAMP-dependent protein kinase activity (Ve/Vo = 2.04). Both casein kinase I and casein kinase II complexes are unstable in 0.5 M NaCl, giving rise by gel filtration under these conditions to the free forms of casein kinase S (Ve/Vo = 2.37, Mr 34 000) and casein kinase TS (Ve/Vo = 2.10, Mr 130 000), respectively. In contrast, the elution volume of cAMP-dependent protein kinase activity is always the same irrespective of the ionic strength of the medium. Casein kinase I, accounting for the whole casein kinase S activity of cytosol, also contains a phosphorylatable 31-kDa protein (p31) which is a substrate of casein kinase S, since its phosphorylation is insensitive to heparin, the heat-stable inhibitor and trifluoperazine, but it is prevented by beryllium. Casein kinase II, on the other hand, apparently results from the association of the whole casein kinase TS (type 2) of rat liver cytosol with a 90-kDa protein substrate (p90) which is distinct from glycogen synthase according to their different peptide mappings. The radiolabelling of p90 is inhibited by heparin, unlabeled GTP and polyglutamates, while it is dramatically and specifically enhanced by polylysine. At least three more protein bands of Mr 58 000, 52 000 and 37 000 are phosphorylated by casein kinase TS in the casein kinase II fraction: their co-elution with casein kinase TS, however, seems to be accidental and their radiolabeling in the presence of polylysine is almost negligible compared to that of p90. It is concluded that p31 and p90 may represent specific targets of casein kinase S and casein kinase TS, respectively, whose intimate association with the enzymes could be functionally significant.