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Polyphosphoinositides have many roles in cell signalling and vesicle trafficking [1–3]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells and rapidly accumulates in hyperosmotically stressed yeast. PI(3,5)P2 is synthesised from PI(3)P in both yeast and mammalian cells [4,5]. A search of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase. Fab1p shows PI(3)P 5-kinase activity both in vivo and in vitro. A yeast strain in which FAB1 had been deleted was unable to synthesise PI(3,5)P2, either in the presence or absence of osmotic shock. A loss of PI(3,5)P2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature. A recombinant glutathione-S-transferase (GST)–Fab1p fusion protein was shown to have selective PI(3)P 5-kinase activity in vitro. Thus, we have demonstrated that Fab1p is a PI(3)P-specific 5-kinase and represents a third class of PIP kinase activity, which we have termed type III. Deletion of the FAB1 gene produces a loss of vacuolar morphology [6]; it is therefore concluded that PI(3,5)P2, the lipid product of Fab1p, is required for normal vacuolar function.

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase are protein-serine/threonine kinases which are important regulators of diverse cellular processes including metabolism, proliferation and differentiation. This study shows that both hypoxia and X irradiation of serum-deprived Chinese hamster V79 cells cause the induction and phosphorylation of the PKC-alpha isoform. The increased induction and phosphorylation of PKC occur mainly in the nuclear fraction. Unlike the PKC activator TPA, neither hypoxic nor radiation stress causes translocation of PKC-alpha from the cytosol to the membrane. The induction of PKC-alpha by hypoxia is accompanied by an increased expression of MAP kinase but, in contrast, this does not occur when PKC-alpha is induced by radiation. Radiation, like TPA, causes a complete redistribution of MAP kinase from the cytosol to the nucleus.

Glycogen synthase preparations which were free of endogenous phosphorylase kinase were phosphorylated to different extents with cyclic-AMP-dependent protein kinase and the phosphopeptides obtained by tryptic digestion were analysed. Limited tryptic digestion of the native enzyme quantitatively released two phosphopeptides (site 1 a and site 1 b) which were soluble in trichloroacetic acid. The trichloroacetic-arid pellet contained a distinct phosphorylation site, which was shown to be identical to the serine residue which is phosphorylated by phosphorylase kinase (site 2). Sites 1 a, 1 b and 2 accounted for virtually all of the phosphorylation of glycogen synthase up to 2.5 molecules phosphate incorporated per subunit. Site 1 a was initially phosphorylated 7–10-fold faster than site 2, and 15–20-fold faster than site 1 b. The phosphorylation of site 1 a became much slower after about 0.5 molecules of phosphate had been incorporated per subunit. Cyclic-GMP-dependent protein kinase also phosphorylated glycogen synthase at sites 1 a, 1 b and 2. The order of phosphorylation was the same as that observed with cyclic-AMP-dependent protein kinase (site 1 a > site 2 > site 1 b). All three sites phosphorylated by cyclic-AMP-dependent protein kinase could be dephosphorylated by protein phosphatase 1. Site 2 was initially dephosphorylated 5–10-fold faster than site la and more than 100-fold faster than site 1 b. The MgATP-dependent protein phosphatase dephosphorylated site 2, site 1a and site 1 b to a very similar manner to protein phosphatase 1. The different rates of phosphorylation and dephosphorylation of the three sites allowed the demonstration to be made that the activity of glycogen synthase depended on the state of phosphorylation of site 2 and site 1 a. The phosphorylation of site 1 b did not influence the activity under the conditions used. Incubation of glycogen synthase with high levels of phosphorylase kinase did not lead to the phosphorylation of either site 1 a or 1 b, or of the three sites phosphorylated by glycogen synthase kinase 3 (sites 3a, 3b and 3c). Similarly no phosphorylation of sites 3a, 3b or 3c by cyclic-AMP-dependent protein kinase was detected. It is concluded that site 2 is the only site at which overlapping substrate specificity occurs between these three glycogen synthase kinases.

The combined application of protein structural techniques, immunochemistry, and molecular biology has permitted an analysis of the differential expression of the genes for the three related protein kinases, C-alpha, -beta, and gamma. The evidence now suggests that the type of protein kinase C in a cell may govern the nature and mechanisms of functional responses.

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.

By use of the autophosphorylated epidermal-growth-factor receptor and the synthetic peptide RRLIE-DAEY(P)AARG, representing an autophosphorylation site of the transforming protein of Rous-sarcoma virus, it is demonstrated that the phosphotyrosyl phosphatase activity of the polycation-stimulated phosphatases is substantially increased by an enzyme-directed effect of ATP or PPi. Concomitant with this increase in phosphotyrosyl phosphatase activity, the phosphorylase phosphatase activity is decreased, thus dramatically changing the substrate specificity of these enzymes. The dephosphorylation of four different phosphotyrosyl sites of the epidermal-growth-factor receptor is neither consecutive nor at random, but a preferred dephosphorylation of the P1 site over the P3 greater than P2 greater than P4 sites is observed. This phosphatase activity represents a substantial fraction of the total phosphotyrosyl phosphatase activity in the post-mitochondrial supernatant of Xenopus laevis oocytes.

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.

The structural analysis of protein kinase C has led to the identification of a family of related gene products. This family of kinases consists of six unique genes that give rise to at least seven polypeptides. The high degree of conservation and the differential distribution of these mRNAs/proteins suggest that they perform distinct functions in vivo. Characterization of the activities of some of these proteins in vitro shows that there are functional differences with respect to both their regulation and substrate specificity. This indicates that each member of this family may play a unique role in signal transduction.

Glycogen synthase can be phosphorylated in vitro by several protein kinases, producing forms of the enzyme that are more dependent on the allosteric activator glucose 6-phosphate (reviewed in [ 1,2]). One of these glycogen synthase kinases is the enzyme cyclic AMP-dependent protein kinase [3,4] which may underlie the inhibition of glycogen synthase that occurs in vivo in response to adrenaline [5,6]. Cyclic AMP-dependent protein kinase has been shown to phosphorylate glycogen synthase on three serine residues in vitro termed site-l a, site-lb and site-2 [7,8]. The initial rate of phosphorylation of site-l a is 7-lo-fold faster than site-2 and 15-20-fold faster than site-lb; the activity of glycogen synthase is determined by the state of phosphorylation of site-2 as well as site-l a [8]. The phosphorylation of site-lb does not appear to have a direct effect on the activity [7,8]. Site-2, which is located seven residues from the N-terminus of glycogen synthase, is the major site phosphorylated by phosphorylase kinase [9,10]. Here, the amino acid sequence surrounding site-la has been determined. This analysis has surprisingly demonstrated that sites-l a and 1 b are separated by only 13 amino acids in the primary structure of glycogen synthase, which comprises -770 residues [ 1 I].

Protein kinase C, the major phorbol ester receptor, was purified from bovine brain and through the use of oligonucleotide probes based on partial amino acid sequence, complementary DNA clones were derived from bovine brain complementary DNA libraries. Thus, the complete amino acid sequence of bovine protein kinase C was determined, revealing a domain structure. At the amino terminal is a cysteine-rich domain with an internal duplication; a putative calcium-binding domain follows, and there is at the carboxyl terminal a domain that shows substantial homology, but not identity, to sequences of other protein kinase.

The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed.

A new family of protein kinase C-related genes has been identified in bovine, human, and rat genomes. The alpha-, beta-, and gamma-type protein kinase sequences are highly homologous, include a kinase domain, and potential calcium-binding sites, and they contain interspersed variable regions. The corresponding genes are located on distinct human chromosomes; the possibility of even greater genetic complexity of this gene family is suggested by Northern and Southern hybridization analyses.

This composite portrait of a “community” of printers reveals the composition of their trade and the unique mixture of businessman and publicist they represented in early America.

Membrane and cytosol fractions from hepatocytes of both normal and streptozotocin-induced diabetic animals were probed with a panel of polyclonal anti-peptide antisera in order to identify protein kinase C (PKC) isoforms. Immunoreactive species were noted with antisera specific for alpha (approximately 81 kDa), beta-II (approximately 82 kDA), epsilon (approximately 95 kDa) and epsilon (approximately 79 kDa). In addition, a species migrating with an apparent size of approximately 94 kDa was also detected in cytosol fractions using an antiserum specific for PKC-alpha. Each of these species was specifically displaced when the PKC-isoform specific peptide was included in the immunodetection system. No immunoreactive species consistent with the presence of the beta-I, gamma, delta and eta isoforms of protein kinase C was observed. Induction of diabetes using streptozotocin invoked selective alterations in the expression of PKC isoforms which were reversed upon insulin therapy. In the cytosol fraction, marked increases of approximately 3-fold occurred in levels of the beta-II isoform and the approximately 90 kDa (upper) form of PKC-alpha, with no apparent/little change in the levels of the approximately 81 kDa (lower) form of PKC-alpha and those of PKC-zeta. Diabetes induction also appeared to have elicited the translocation of PKC-beta-II and the approximately 81 kDa (lower) form of PKC-alpha to the membrane fraction where immunoreactivity for these species was now apparent. The level of PKC-epsilon, which was noted only in membrane fractions, was also increased upon induction of diabetes. It is suggested that the selective alterations in the expression of PKC isoforms occurring upon streptozotocin-induced diabetes may lead to altered cellular functioning and underly defects in inhibitory G-protein functioning and insulin action which characterise this animal model of diabetes.

The protein kinase C (PKC) family participates in a ubiquitous cell signalling system utilizing increased turnover of phosphoinositides. Because down-regulation of total PKC activity has been implicated in the acquisition of a morphologically differentiated phenotype in SH-SY5Y neuroblastoma cells, we aimed to identify the specific PKC isoforms in this process. Here we report that intracellular delivery of PKC-alpha and -epsilon, but not -beta, -gamma or -delta isoform-specific antibodies is sufficient to induce acquisition of a morphologically differentiated phenotype in SH-SY5Y neuroblastoma cells.

Threonine and tyrosine residue phosphorylation of a 42 kDa protein identified as mitogen-activated protein kinase (MAP kinase) was stimulated in extracts from TPA-pretreated cells. It is further shown that TPA pretreatment leads to the enhancement of an activity that will induce reactivation of dephosphorylated/inactivated MAP kinase. This TPA-induced activity induces the threonine and tyrosine phosphorylation of p42 in extracts from unstimulated cells.

Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. One important mediator of sensitization is bradykinin (BK), a peptide released as a consequence of tissue damage. BK enhances the membrane ionic current activated by heat in nociceptive neurons, using a pathway that involves activation of protein kinase C (PKC). We find that five PKC isoforms are present in sensory neurons but that only PKC-epsilon is translocated to the cell membrane by BK. The heat response is sensitized when constitutively active PKC-epsilon is incorporated into nociceptive neurons. Conversely, BK-induced sensitization is suppressed by a specific peptide inhibitor of PKC-epsilon. We conclude that PKC-epsilon is principally responsible for sensitization of the heat response in nociceptors by bradykinin.

The purpose of the present study was to examine protein kinase C (PKC) isotype expression in T lymphoblasts derived from peripheral blood and the T leukaemic cell Jurkat. Using antisera reactive with PKC alpha, beta 1, and beta 2 and gamma, it was observed that T cells expressed two PKC isotypes, PKC alpha and beta 1. No PKC gamma was detected in T lymphocytes. In lymphoblasts, high levels of PKC beta compared to PKC alpha were found whereas Jurkat cells expressed high levels of alpha compared to PKC beta. Differences in the calcium sensitivity of phorbol ester-induced phosphorylation were observed in Jurkat and T lymphoblasts which correlated with the relative levels of PKC alpha and beta isotypes expressed by the cells.

The substrate specificity of purified PKC-α, -β and -γ has been investigated. A series of synthetic peptides based upon the sequence surrounding serine-7 in glycogen synthase were generated and used to determine the basic residue requirements of these PKC isotypes. While PKC-α and -β are indistinguishable in their phosphorylation of these peptides, PKC-γ shows a distinct specificity profile for these synthetic substrates.

Nerve growth factor (NGF) treatment of PC12 cells induced a 2.8-fold increase in protein kinase C activity concomitant with differentiation and acquisition or neurites. PKC protein isoforms were separated by sequential chromatography on DEAE-Sephacel/hydroxylapatite. A broad peak or PKC activity eluted which corresponded to the alpha PKC isoform. In control cells, message for all six PKC isoforms was detected and expressed as epsilon>zeta=gamma>delta>beta>alpha. Western blot or whole cell lysates revealed a large increase in the beta11, while slight changes were observed for the other five PKC isoforms during treatment (12-14 days) with NGF (50 ng/ml). In parallel, coordinate changes in the expression of the individual transcripts for the six isoforms occurred during NGF treatment. Induction and accumulation of PKC beta11 may play a role in maintenance or neuronal morphology.

The mammalian Ptdlns 3-kinase is shown to be inhibited by low nanomolar concentrations of demethoxyviridin, an antifungal agent structurally related to wortmannin. The inhibitory potency of both compounds could be observed in purified Ptdlns 3-kinase whether or not the regulatory subunit (p85α) was present, suggesting that the inhibitors bind to the catalytic subunit (p110) of the Ptdlns 3-kinase. These inhibitors also show similar potency against the intrinsic p85-phosphorylating activity of the p110-kinase. However, the structurally related Ptdlns 3-kinase from Saccharomyces cerevisae (Vps34p) is not inhibited by either compound. Both inhibitors target the mammalian Ptdlns 3-kinase in vitro and in vivo, implying that these compounds should be useful in suppressing Ptdlns 3-kinase in mammalian systems. The inhibitors did not affect the mammalian Ptdlns 4-kinase, but they are able to inhibit a membrane-associated Ptdlns 4-kinase from Schizosacchromyces pombe.

The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) in a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRK1) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified ‘PKC’ sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.

The epidermal growth factor (EGF) receptor is a tyrosine-specific protein kinase with autophosphorylating activity1–4. A 300 amino acid-long region of the receptor's cytoplasmic domain matches (35–90 % homology) sequences of transforming proteins from the src family5 and includes a putative nucleotide binding site6. Several of the src transforming proteins have tyrosine kinase activity7, but v-erb-B, which appears to be a truncated EGF receptor, is virtually identical to the receptor over this region and yet lacks detectable kinase activity8,9. To locate possible acceptor sites in the v-erb-B protein, we have mapped these sites in the human EGF receptor. We report here that three tyrosine sites near the C-terminus are phosphorylated in vitro. In intact cells, we find that EGF stimulates phosphorylation of several sites, the tyrosine 14 residues from the C-terminus being modified the most extensively. The equivalent site is absent in the v-erb-B protein of avian erythroblastosis virus (AEV) and may influence tyrosine kinase activity10,11.

A kinase defective mutant of PKC-α down-regulates in response to phorboI esters as effectively as the wild-type protein when introduced into COS-1 cells. This demonstrates that intramolecular autophosphorylation is not a prerequisite for down-regulation.

Phosphoinositide 3-kinase, an enzyme that is known to transduce signals received by a variety of receptor types, has been found to mediate agonist-dependent membrane ruffling via the small GTP-binding protein Rac.

A novel member of the inositol lipid-specific phospholipase C family, PtdIns-PLCβ3, is shown to be activated by βγ subunits of the heterotrimeric GTP-binding protein, transducin. The activation is a direct effect since it is observed with the purified proteins. Furthermore, the activation is blocked by the GDP-liganded α subunit of transducin, confirming that the effect is due to free βγ subunits. The implications with respect to receptor-PtdIns-PLC coupling are discussed.

A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 mumol.min-1.mg-1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.

The calcium- and phospholipid-dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE-cellulose chromatography, gel filtration and hydrophobic chromatography on octyl-Sepharose and phenyl-Sepharose. The phorbol ester receptor co-purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS-polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6-2 and 35-80 micrograms/ml). These apparent Ka values were reduced 2- to 3-fold by either diolein (20 micrograms/ml) or phorbol-12,13-dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol-12,13-dibutyrate ( [3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine-dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.

Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE-GEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the levels of immunoreactive polypeptide and extractable calcium- and phospholipid-dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13-dibutyrate protein kinase C was essentially undetectable by 40 hours; the half-life of this down-regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium- and phospholipid-dependent kinase activity precisely parallels the phorbol ester induced down-regulation of binding and responsiveness in Swiss 3T3 cells.

The transforming protein of the Abelson murine leukaemia virus encodes a protein-tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation.

As a means of determining the role of protein kinase C in the signal transduction from novel growth factors and hormones, we investigated the effects of well-characterized agents on the phosphorylation state of protein kinase C itself. These studies show that agents that stimulate protein kinase C either directly (phorbol esters) or indirectly through phosphatidylinositol breakdown (platelet-derived growth factor) induce an increase in the phosphorylation state of the kinase. By contrast, epidermal growth factor, which does not stimulate protein kinase C in fibroblasts, does not increase the phosphorylation state of protein kinase C, but leads to a decrease. The data suggest that the phosphorylation state of protein kinase C is dynamically controlled and can be used to provide evidence of protein kinase C activation.

The phorbol-ester-induced loss of protein kinase C that has been documented in many cell types appears to be a critical event in the generation of a cellular refractory state. We have investigated here the synthesis and degradation of the protein kinase C polypeptide in order to determine why its steady-state amounts are depleted in response to phorbol esters. These results indicate that depletion is due to an increased rate of degradation, with no change either in mRNA amounts or in rates of polypeptide synthesis.

Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.

It is generally assumed that branched chain 2-oxoacids formed from leucine, isoleucine and valine are oxidised by dehydrogenase complex(es) analogous to pyruvate and 2-oxoglutarate dehydrogenase complexes. There is much recent work on the regulation of oxidation of branched chain amino acids and 2-oxo-acids in vivo but little information on substrate kinetics or regulation by metabolite effectors of the branched chain 2-oxo-acid dehydrogenase complex(es). We describe here a method for extraction and partial purification of a complex from rat liver mitochondria, K m values for substrates and inhibition by isovaleryl CoA (competitive with CoA) and NADH (competitive with NAD÷). Evidence is given that a single complex may oxidise all 3 branched chain 2-oxo-acids.

Clinical pharmacists can deliver safer and more efficacious drug therapy to end-stage renal patients with knowledge and application of the pharmacokinetics of haemodialysis. The physical, chemical and pharmacokinetic properties of a drug will determine its degree of dialysability. These properties included water solubility, molecular weight, protein binding, volume of distribution, and the fraction, route and rate of drug elimination. Various dialysis-induced metabolic alterations must also be considered. With an understanding of these factors, the pharmacist can determine whether dosage adjustments are necessary and, if so, make the required calculations accurately.