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.

The protein kinase C (PKC) family consists of 11 isoenzymes that, due to structural and enzymatic differences, can be subdivided into three groups: The Ca 2+ -dependent, diacylglycerol (DAG)-activated cPKCs (conventional PKCs: a, b 1 , b 2 , g); the Ca 2+ -independent, DAG-activated nPKCs (novel PKCs: d,1, h, u, m), and the Ca 2+ -dependent, DAG non-responsive aPKCs (atypical PKCs: z, l/i). PKCm is a novel PKC, but with some special structural and enzymatic properties.Keywords: protein kinase Cd; activation; autophosphorylation; down-regulation; structure; tyrosine phosphorylation; function.Protein kinase Cd (PKCd) is the most thoroughly studied member of the nPKC subfamily. After the discovery of the enzyme in 1986, its cloning in 1987, and its first purification to homogeneity in 1990, several groups have focused their interest on this PKC isoenzyme and have reported on its expression, structural and enzymatic properties, and cellular functions. Some information has accumulated on the mode and structural requirements of activation and down-regulation of PKCd. Recently, the role of phosphorylation of PKCd, i.e. either autophosphorylation or phosphorylation by an exogenous protein kinase, for the regulation of its enzymatic activity could to some extent be elucidated and compared to that of other PKC isoforms. However, many questions remain to be answered. The same holds true for PKCd-specific substrate phosphorylation and biological functions. Various more or less useful methods have been applied to elucidate PKCd-specific functions in a given cell. Data accumulated that indicate a role of PKCd in growth inhibition, differentiation, apoptosis, and tumor suppression. However, the knowledge on PKCd-specific substrate phosphorylation resulting in these specific cellular effects is as yet extremely poor. The PKC familyProtein kinase C (PKC) was initially identified and characterized as a proteolytically activated kinase called protein kinase M [1,2]. The proenzyme (PKC) was then shown to exhibit calciumand phospholipid-dependent kinase activity independently of proteolytic activation [3]. In the presence of diacylglycerol (DAG), a well-known second messenger molecule [4,5], the calcium concentration necessary for stimulation of the enzyme was reduced to physiological levels [6]. DAG was known to be produced by phosphatidyl inositol turnover induced by cellular receptors upon interaction with numerous extracellular factors [4,5]. This indicated that PKC might play an important role in transmembrane signal transduction. Another key discovery was that PKC was the major intracellular receptor for the tumorpromoting phorbol esters, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) [7,8]. When TPA was used as PKC activator in vivo, PKC proved to be linked to the signal-induced modulation of a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis, and tumor development. In 1986, the cDNAs of three PKC isoenzymes (a, b, g) were cloned [9±11]. The PKCb gene was found to generate two isoe...