Isozymic forms of rat brain Ca2+-activated and phospholipid-dependent protein kinase.

Abstract

Three forms of rat brain Ca2+-activated and phospholipid-dependent protein kinase (EC 2.7.1.37) were separated by hydroxylapatite column chromatography. These enzymes, designated type I, II, and m protein kinase C, all have a molecular weight of 82,000, undergo autophosphorylation in the presence of Ca2', phosphatidylserine, and diolein, and bind [3H]phorbol 12,13-dibutyrate. Autophosphorylation of these kinases resulted in an incorporation of 1-1.5 mol of 32p per mol of enzyme. Two-dimensional peptide mapping analysis revealed that these kinases had different sites of autophosphorylation. Phosphoamino acid analysis showed that type I and type m protein kinase C primarily autophosphorylated at a serine residue, whereas type II kinase autophosphorylated at both serine and threonine residues. In addition, polyconal antibodies raised against a mixture of three types of the kinase preferentially inhibited type I and type II enzymes. Monoclonal antibodies against type I and type II kinase only recognized their respective enzymes but not the type m enzyme. These results demonstrate the presence of isozymic forms of protein kinase C in rat brain.The Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) was first found in rat brain as a proteolytically activated protein kinase (1, 2). This enzyme was later shown to require Ca2+ and phospholipid (3,4) and to be further activated by diacylglycerol (DAG) (5), which markedly increased the affinity of the kinase for both Ca2+ and phospholipid. Protein kinase C has been purified to near homogeneity from many sources (6-12); however, the presence of isozymic forms of the enzyme has not been reported. This kinase is a monomeric polypeptide of molecular weight 82,000 that appears to be composed of two functionally different domains: one is a hydrophobic domain that may bind to membranes, and the other is a hydrophilic domain that carries the catalytically active center. These two domains are cleaved by Ca2+-dependent neutral proteases to produce a Ca2+-and phospholipid-independent active enzyme fragment (13). Intracellular stimulation of protein kinase C can be effected by the DAG generated from the signal-induced breakdown of inositol phospholipids or by the addition of synthetic DAG or tumor-promoting phorbol esters (14,15).Protein kinase C is an ubiquitous enzyme found in a variety of mammalian tissues (16,17). This enzyme has attracted particular attention because it plays a pivotal role in controlling many cellular functions. In addition, this enzyme has been identified as a putative receptor for tumor-promoting phorbol esters (18, 19). Recently we have prepared polyclonal and monoclonal antibodies against rat brain protein kinase C. In the process of characterizing these antibodies we noticed that some purified kinase preparations were less sensitive to inhibition by these antibodies. Therefore, we refined our purification procedure to separate the different forms ofprotein kinase C. In this report, we provide evidence to demonstrate the exist...

Protein kinase C during differentiation of human promyelocytic leukemia cell line, HL‐60

“…Initially, four cDNA clones, dyt ,&I, &IBfE and y, were isolated, which are highly conserved among various mammalian species. PKC from the brain tissue is normaBy resolved into three subfractions, types I, II and III, upon hydroxyapatite column chromatography [3], which correspond to the enzymes encoded by y-, P-@I-plus #II-) and CY-cDNA clone, respectively [4]. Recently, three additional cDNA clones of PKC, 6, 6 and 1, have been isolated from the rat brain cDNA library {S] (also for a review, see [l]).…”

Distinct functions of down‐regulation‐sensitive and ‐resistant types of protein kinase C in rabbit aortic smooth muscle cells

“…Subsequently, three different protein molecules of this enzyme have been purified from rat brain [8]. The different types of the purified enzyme show very similar catalytic, kinetic and physical properties but are distinguishable from one another by their respective specific antibodies [8].…”

Isoenzyme patterns of protein kinase C and a phospholipid‐dependent but Ca2+‐inhibited enzyme fraction in the crude extracts of different tissues

“…Beside the four subspecies found previously in the mammalian brain, three additional members of rat protein kinase C family have also been described [7][8][9]. In the brain three major types of protein kinase C isoenzymes have been resolved by hydroxyapatite chromatography [10][11][12] and the correspondence of type I, II and III isoenzymes to the cDNA clones designated 7, 13x+/~ II and a, respectively, has been determined [13]. Protein kinase C isoenzymes are differentially distributed in various tissues [6,9,11,14,15] and the subspecies show a slightly different mode of activation, kinetic properties and substrate specificities [9,12,16,17], suggesting different functional roles.…”

“…The three isoenzyme types mentioned have been separated by hydroxyapatite chromatography from purified or at least partially purified preparations obtained from brain [10][11][12]. In the course of purification the recovery of the isoenzyme fractions may be different.…”