The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.

The granule cell-enriched Ca2+/calmodulindependent protein kinase (CaM kinase-Gr) is a recently discovered neuron-specific enzyme. The kinase avidly phosphorylates synapsin I and contains a polyglutamate sequence, which suggests an association with chromatin as well. A possible role in synapsin I phosphorylation and in nuclear Ca2+ signaling was supported by immunochemical and ultrastructural examination of CaM kinase-Gr distribution. CaM kinase-Gr immunoreactivity was present in the molecular and granule cell layers of the rat cerebellum. This pattern corresponded to the occurrence of the enzyme in the granule cell axons and nuclei, respectively. Immunoblots confirmed these findings. Thus, CaM kinase-Gr may mediate and coordinate Ca2+-signaling within different subcellular compartments.Ca2+/calmodulin-dependent protein kinases (CaM kinases) have been implicated in neuronal communication by regulating neurotransmitter biosynthesis, neurotransmitter release, alterations in cytoskeletal components, and possible regulation of gene expression (1-5). A brain-specific CaM kinase has recently been isolated and purified and part of its encoding nucleotide sequence has been cloned (6). This kinase shows certain catalytic and regulatory similarities to CaM kinase II but exhibits a number of unique characteristics, including amino acid sequence, subunit organization, subcellular distribution, and immunohistochemical localization. Because this kinase protein is enriched in the granule cells of the cerebellar cortex, it has been called the granule cell-enriched CaM kinase (CaM kinase-Gr). The enzyme has been purified to homogeneity and consists of two polypeptides with apparent Mr values of 65,000 and 67,000 (6). Partial cDNA sequence data indicate that mouse and human brains contain homologues to the rat enzyme that, nevertheless, exhibit considerable divergence in their nucleotide sequence (7,8).The potential physiological roles of CaM kinase-Gr depend on its substrate specificity and its subcellular availability. This kinase phosphorylates synapsin I on the head and tail domains (6) and may thereby promote neurotransmitter release by analogy to the action of CaM kinase II (9, 10). Moreover, CaM kinase-Gr contains a polyglutamate-rich sequence (6), which characterizes several chromatinassociated proteins (11). Thus, CaM kinase-Gr may regulate different neuronal reactions in different subcellular compartments. The objective of this study was to determine the subcellular compartments in which CaM kinase-Gr occurs.MATERIALS AND METHODS Primary Antiserum. Rabbit antibodies were raised against a f3-galactosidase fusion product of CaM kinase-Gr expressed in and purified from Escherichia coli. The antiserum was affinity-purified by adsorption to mammalian CaM kinase-Gr isolated from rat cerebellum (6). The resulting monospecific antibody preparation was employed throughout this study.Immunoblots. Punches from the molecular layer and granule cell layer of cerebellar vermis of adult rats (0.1 mg of each tissue) were susp...

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 40C as well as at 30'C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase I, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase U by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.Phorbol esters influence cellular functions at various levels (1, 2), presumably by activating protein kinase C (3, 4). Some cellular effects can be detected rapidly after the addition of phorbol esters. These effects include the regulation of ionic transport, release of bioactive substances, and receptor down-regulation (1,2,5). Other effects of phorbol esters target the genome, resulting in the regulation of DNA replication or in the modulation of gene expression. Examples of genes that appear to be induced by phorbol esters include ornithine decarboxylase in epidermal cells (6,7), interleukin 2 and IL-2 receptor in mouse T cells and Tlymphoma cells (8-10), c-fos in U937 monocytes and in HL-60 cells (11), actin and vimentin in K562 erythroleukemia cells (12), and calcitonin in thyroid medullary carcinoma cells (13). Genes that seem to be repressed by phorbol esters include globin in Friend erythroleukemia cells (14), glycophorin in K562 erythroleukemia cells (15), and c-myc in thyroid medullary carcinoma cells (13). Moreover, phorbol esters increase the frequency of initiation of DNA replication for bacteriophage X injected into Xenopus eggs (16).The effects of phorbol esters on gene transcription generally require more time to become manifest than the effects on ionic fluxes or receptors. Thus, the regulation of c-fos mRNA in U937 monocytes is maximal after 30 min (11) and that of glycophorin and actin genes becomes evident after about 1 hr (12, 15), whereas the effect on c-myc mRNA is observed 4 hr after exposure of the cells to the tumor promoter (13). The relative delay in these responses may reflect an indirect effect ofphorbol esters on gene transcription, requiring one or more intermediary steps or a cascade of reactions; alternatively, phorbol esters may alter gene transcription more directly by stimulating protein kinase C, which then may phosphorylate a polypeptide(s) involved in transcriptional regu...

The effect of phorbol esters on the extent of phosphorylation of. receptors for insulin and somatomedin C (insulin-like growth factor I) was studied in intact IM-9 cells that were labeled by incubation with H332P04. The. tumor-promoting phorbol esters phorbol tetradecanoate acetate (TPA) and phorbol dibutyrate, but not the inactive-4a-phorbol, enhanced phosphorylation of the ,B subunit of both receptors approximately 4-fold; 70 nM TPA maximally stimulated phosphorylation of both receptors, -whereas concentrations less than or equal to 0.7 nM had no observable effect. Insulin also enhanced the phosphorylation of the 13 subunit of the insulin receptor, and its effects appeared to be additive to those of TPA. Peptide maps indicated that at least some of the residues phosphorylated by these two agents are distinct. These results suggest a possible role of protein kinase C in regulating insulin and somatomedin C receptors.Exposure of intact cells to tumor-promoting phorbol diesters results in a profound decrease in their affinity for insulin (1, 2). The rapidity of this response, which occurs within 5 min, suggests that it may be a relatively proximate response to phorbol esters. Because phorbol esters bind to and directly activate protein kinase C (3, 4), they may affect insulin binding by stimulating protein kinase C-induced phosphorylation of insulin receptors. To test this hypothesis, we investigated the effect of phorbol esters on the extent and pattern of phosphorylation of insulin receptors. Because of the extensive similarities between insulin and somatomedin C (insulin-like growth factor, type I) receptors, both with respect to their structure and their mode of regulation, the effects of phorbol esters on somatomedin C receptor phosphorylation were also studied. METHODS aIR-i is a monoclonal antibody to the insulin receptor. aIR-3 is a monoclonal antibody to the somatomedin C receptor. A410 is a rabbit anti-insulin receptor antiserum. The production and properties of these antibodies have been described (5-7).Cell Labeling. IM-9 cells (a human B-lymphocyte line) were cultured at a density of 105-106 cells per ml of RPMI 1640 medium containing 10% fetal calf serum; 109 cells were washed twice with phosphate-free RPMI 1640 medium, resuspended in 40 ml of phosphate-free RPMI medium containing 20 mM Hepes (pH 7.4) and 4 mCi of H332PO4 (1 Ci = 37 GBq), and incubated for 1 hr at 37°C. The cells were then divided into aliquots, which were incubated with no additions, phorbol derivatives, or insulin as indicated in the figure legends. The incubations were stopped by diluting the cells with 4 vol of icecold phosphate-buffered saline containing 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 4 mM EDTA, and 0.2 mM sodium vanadate. The cells were washed twice with this buffer.Immunoprecipitation and NaDodSO4/Polyacrylamide Gel Electrophoresis. The washed labeled cells were solubilized with 1% Triton X-100 in 50 mM Tris-HCI (pH 7.7) containing 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 0.2 mM sodium...

Phorbol esters are potent tumour-promoting agents that exert pleiotropic effects on cells. Among these are the control of growth, stimulation of release of stored bioactive constituents and regulation of growth-factor surface receptors. Phorbol esters bind to and activate protein kinase C, leading to the phosphorylation of specific protein substrates presumed to be necessary for eliciting the full response. Strong evidence exists that specific binding of tumour promoter occurs at the membrane level in intact cells, resulting in activation of protein kinase C. Recent evidence concerning the release of bioactive constituents from platelets and neutrophils has linked agonist-induced protein kinase C activation and Ca2+ mobilization in a synergistic mechanism. Here we present a novel model of synergism between Ca2+ and phorbol esters that leads to transferrin receptor phosphorylation and down-regulation in HL-60 human leukaemic cells. Raising intracellular Ca2+, although ineffective by itself, increases the potency and rate of action of phorbol ester for activating protein kinase C and mediating transferrin receptor phosphorylation and down-regulation. We propose a molecular model in which increased intracellular Ca2+ recruits protein kinase C to the plasma membrane, thus "priming' the system for activation by phorbol ester.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells (CaM kinase Gr) is a neuronal calmodulin-dependent protein kinase whose purification and partial cloning from rat brain has been described. A combination of the polymerase chain reaction and cDNA library screening was used to determine the DNA sequence that encodes most of the remaining polypeptide sequence. The deduced amino acid sequence was confirmed by comparison with the peptide sequence from purified CaM kinase Gr. Analysis of this sequence indicated the presence of potential catalytic, regulatory, and association domains with 42% overall homology tothe ct subunitofanother neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase II. The degree of homology within the catalytic domain was 58% with conservation of all invariant amino acids. The portion of sequence that extended from the hypothesized calmodulin-binding domain to the carboxyl terminus of the protein was identical at both the amino acid and nucleotide level to the noncatalytic, calmodulin-binding protein calspermin from rat testis. Screenmg a genomic library with a portion of the cDNA for CaM kinase Gr allowed the isolation of a genomic clone that contained at least 9 kilobases (kb) of the gene for CaM kinase Gr. Analysis of the sequence revealed that the coding sequences for calspermin were contained within the CaM kinase Gr gene and that alternative splicing of internal exons may lead to the formation of the two different proteins, CaM kinase Gr and calspermin.

Neuronal differentiation not only entails the acquisition of various molecular and cellular characteristics but also involves the loss of characteristics that appear only during certain stages of development (1)(2)(3)(4). Calcium ions have been implicated in the regulation ofneuronal development (5-7) and Ca2+/calmodulin-dependent protein kinases have been demonstrated to be an important pathway for Ca2+ signaling (8,9). In this report we describe the developmental pattern ofexpression ofgranule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr), which is comprised of Mr 65,000 and 67,000 polypeptides and has been localized to various neuronal populations (10). In the adult cerebellum, this enzyme was found to be concentrated in granule cells but absent from Purkinje cells (10). Because the development and circuitry of these cells have been extensively studied, we attempted to correlate the production of CaM kinase-Gr with different stages of cerebellar development. The conclusions arrived at in these studies were subsequently extended by examining the appearance of CaM kinase-Gr in the hippocampus. The present observations of developmental expression of CaM kinase-Gr typify the acquisition and loss of a Ca2+-signaling pathway during development.MATERIALS AND METHODS Primary Antiserum. Rabbit antibodies were raised against a f-galactosidase fusion product of CaM kinase-Gr expressed in and purified from Escherichia coli. The antiserum was affinity-purified by adsorption to mammalian CaM kinase-Gr isolated from rat cerebellum (10). The resulting monospecific antibody preparation was employed throughout this study.Immunoblots. Cerebella from postnatal day (PND)-3 and adult rats were homogenized with 10 volumes of buffer containing 25 mM Hepes (pH 7.5), 2 mM EDTA, 0.1 mg of phenylmethylsulfonyl fluoride per ml, and 20 ,ug of leupetin per ml. Equivalent amounts of homogenate were electrophoresed in duplicate in SDS/10% polyacrylamide gels (11) and the proteins were electroblotted onto nitrocellulose paper (12). Blots were incubated at 40C for 16 hr in 25 mM Tris HCl, pH 7.5/0.15 M NaCI/1 mg of polyethylene glycol 20,000 per ml/3 mg of bovine serum albumin per ml. The blots were subsequently incubated with the affinity-purified antibody to CaM kinase-Gr in the same buffer for 1 hr at room temperature followed by extensive washing. Control blots were similarly processed but without the primary antibody. Blots were incubated together with alkaline phosphatase conjugated to goat anti-rabbit IgG for 1 hr at room temperature followed by extensive washing. Immunoreactive material was visualized by the addition of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (13).Immunohistochemistry. Animals at various ages from embryonic day (E)-22 to PND-14 (where PND-0 is the day of birth) were deeply anesthetized with pentobarbital (100 mg/ kg) and sacrificed by cardiac perfusion with saline followed by 4% paraformaldehyde. The brains were removed, postfixed, and immersed in 20% sucrose. Bra...