No abstract

Various inhibitors were tested for their potential to suppress the kinase activity of protein kinase C tt (PKC~t) in vitro and in vivo. Among the staurosporine-derived, rather selective PKC inhibitors the indolocarbazole G6 6976 previously shown to inhibit preferentially cPKC isotypes proved to be a potent inhibitor of PKC~t with an IC5o of 20 nM, whereas the bisindolylmaleimide G6 6983 was extremely ineffective in suppressing PKCtt kinase activity with a thousand-fold higher ICso of 20 ~M. Other strong inhibitors of PKC~t were the rather unspecific inldbitors staurosporine and K252a. Contrary to the poor inhibition of PKCtt by G~ 6983, this compound was found to suppress in vitro kinase activity of PKC isoenzymes from all three subgroups very effectively with IC5o values from 7 to 60 nM. Thus, Gii 6983 was able to differentiate between PKC~t and other PKC isoenzymes being useful for selective determination of PKC~t kinase activity in the presence of other PKC isoenzymes.

Native protein kinase C_~ from porcine spleen is phosphorylated in vitro by the tyrosine kinase src and to a much smaller extent by fyn. The tyrosine phosphorylation of PKC~ is restricted to the activated state of the enzyme, i.e. it occurs only in the presence of an activator, such as TPA or bryostatin. Upon phosphorylation at tyrosine, the apparent molecular weight of PKC~ increases by 6 kDa. Phosphorylation by src induces a stimulation of PKC~ activity apparently exhibiting some substrate selectivity. Other PKC isoenzymes, such as cPKC (g~,7), are not phosphorylated by src or only to a very small extent. This phosphorylation is not dependent on TPA and does not cause an increase in activity and molecular weight of the enzyme.