Immunodeficiency in protein kinase cbeta-deficient mice.

Cross-linking of the antigen receptor on lymphocytes by antigens or antibodies to the receptor results in activation of enzymes of the protein kinase C (PKC) family. Mice homozygous for a targeted disruption of the gene encoding the PKC-betaI and PKC-betaII isoforms develop an immunodeficiency characterized by impaired humoral immune responses and reduced cellular responses of B cells, which is similar to X-linked immunodeficiency in mice. Thus PKC-betaI and PKC-betaII play an important role in B cell activation and may be functionally linked to Bruton's tyrosine kinase in antigen receptor-mediated signal transduction.

The complete primary structure of protein kinase C--the major phorbol ester receptor.

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.

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells.

BACKGROUND: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3 alpha, GSK-3 beta and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3 alpha and GSK-3 beta can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3. RESULTS: Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3 alpha or GSK-3 beta decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies. CONCLUSIONS: Our data indicate that GSK-3 alpha and/or GSK-3 beta, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.

Signalling from TPA to MAP kinase requires protein kinase C, raf and MEK: reconstitution of the signalling pathway in vitro.

The phorbol ester PMA/TPA (phorbol 12-myristate 13-acetate) is a potent tumor promoter which mimics distinct intracellular signalling events triggered by activated growth factor receptors, e.g. the activation of MAP kinases. The largest known family of TPA-binding proteins comprise members of the protein kinase C (PKC) family although other TPA-binding proteins outside the PKC family have recently been identified. In this report we addressed the mechanism and the pathway by which TPA induces the activation of MAPkinases. Using recombinant proteins and in vitro phosphorylation reactions we identified the components in the signal transduction pathway from TPA to MAPkinase and we show that the activation of MAPkinase by TPA requires the presence of protein kinase C, c-raf and the MAPkinase activator MEK. We also find that the activation of raf autophosphorylation in vitro correlates with the ability of Raf to signal to MAPkinase. Thus the activation of Raf by PKC apparently can trigger the same signalling pathway as oncogenic Raf or Raf activation by ras in combination with tyrosine phosphorylation.

Kinase activities of c-Mos and v-Mos proteins: a single amino acid exchange is responsible for constitutive activation of the 124 v-Mos kinase.

The Mos protein kinase is a serine-/threonine-specific protein kinase with a crucial role in meiotic cell divisions in vertebrates. Several oncogenic derivatives of the c-Mos protein have been discovered in murine retroviruses. These proteins have acquired mutations and exhibit different degrees of protein kinase activity in vitro. In an attempt to understand the factors governing Mos protein kinase activity we have compared the kinase activities of the wild-type c-Mos protein and two v-Mos proteins (strain HT1 and MSV124) after expression in insect cells. Only the 124 v-Mos protein showed kinase activity in vitro as measured by autophosphorylation, vimentin phosphorylation or by phosphorylation and activation of MAP kinase kinase. By domain swapping and site-directed mutagenesis we identified a single point mutation in the 124 v-Mos protein (Arg145-->Gly) which is responsible for its constitutive activity. This residue is located in the alpha-helix C of the kinase domain close to the ATP binding fold and is conserved in all known c-Mos proteins. Introduction of the corresponding mutation into HT1 v-Mos and into murine c-Mos activated both proteins for autophosphorylation, vimentin phosphorylation and for signalling via MAP kinase kinase in vitro. We hypothesize that the Arg145-->Gly mutation found in 124 v-Mos mimicks a conformational change which might be an obligatory step in the activation of c-Mos in vivo.

Evidence for different signalling pathways of PKC zeta and ras-p21 in Xenopus oocytes.

Considerable effort has been devoted to identifying critical steps in mitogenic signal transduction pathways. Recently, the atypical PKC zeta isoform has attracted great interest since it has been reported to induce GVBD in Xenopus oocytes and transformation of NIH3T3 fibroblasts, two processes closely linked with the regulation of cell division. Furthermore, PKC zeta has been proposed as an essential effector for ras-p21 function and therefore may be an essential component of the signalling pathway(s) activated by mitogens. In this study we have analysed the responses induced in Xenopus oocytes after microinjection of purified recombinant PKC zeta protein. Microinjection of PKC zeta induced the early activation of MPF which precedes GVBD and also induced the activation of MAP kinase and S6 kinase II. The activation of MPF, MAP kinase and S6 kinase II by PKC zeta was sensitive to cycloheximide, while induction of GVBD was independent of protein synthesis. These results indicate that PKC zeta induces the activation of at least two pathways, only one of them leading to the activation of MAP kinase. By contrast, neither the induction of GVBD nor the activation of MPF, MAPK and S6 kinase II induced by the ras-p21 protein were dependent on protein synthesis. Thus, the comparison of these responses suggests that PKC zeta most likely does not mediate the ras-induced signal transduction pathway in Xenopus laevis oocytes.

Activation of the c-Raf protein kinase by protein kinase C phosphorylation.

The product of the c-raf-1 proto-oncogene is a cytoplasmic serine/threonine protein kinase that appears to be activated in signal transduction from a variety of cell-surface receptors. The mechanism of c-Raf activation upon stimulation of cell-surface receptors is not clear, but there seem to exist multiple pathways of activation which involve tyrosine and/or serine phosphorylation of the c-Raf protein in vivo. The activated state of Raf is reflected in an increased apparent molecular weight of the Raf protein in sodium dodecyl sulfate-polyacrylamide gels owing to hyperphosphorylation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) is one of the agents able to induce this hyperphosphorylation of Raf in vivo, suggesting that protein kinase C (PKC) may be involved in the activation of c-Raf in particular situations. Using recombinant baculoviruses expressing PKC and Raf polypeptides, we show here that conventional PKC types (alpha, beta, gamma) but not novel types (delta, zeta, eta) or the unrelated Mos kinase are able to activate c-Raf in a TPA-dependent manner upon coexpression in insect cells. Direct phosphorylation of the Raf protein with PKC in vitro also enhanced the kinase activity of c-Raf, suggesting that c-Raf acts immediately downstream of PKC in a protein kinase cascade which is triggered by TPA and may lead to transcriptional activation of TPA-inducible genes and tumor promotion.

Protein kinase C.

Based on the molecular structure of the individual members of the protein kinase C family, general properties and the mode of activation of this enzyme family are discussed. Examples are presented of how the investigation of protein kinase C function in vivo has been approached at the molecular level.

Expression of a functional protein kinase C-gamma using a baculovirus vector: purification and characterisation of a single protein kinase C iso-enzyme.

The cloning of complementary DNAs for protein kinase C (PKC) has revealed a multi-gene family of closely related protein kinases [Parker et al. (1986) Science 233, 853-859; Coussens et al. (1986) Science 233, 859-866]. In vivo, the distribution of the PKC isoenzymes follows a fairly tissue-specific pattern suggesting that functional differences exist between the members of this kinase family. To initiate a detailed characterisation of the individual isoenzymes, and as an alternative approach to purifying and separating the individual PKC types and their splice variants from mammalian tissues, we have expressed the bovine PKC type gamma in insect cells using a baculovirus expression vector. The bovine protein constitutes one of the major proteins in infected cells and can be purified to near homogeneity by a 2-step procedure. Analysis of the purified protein confirms that it has authentic mammalian PKC characteristics with respect to phospholipid dependence and phorbol ester binding. The bovine PKC gamma purified from infected cells is post-translationally modified and resolves into a doublet of molecular weights 82,000 and 84,000 upon SDS-polyacrylamide gel electrophoresis. These two size classes of polypeptides appear to result from differential phosphorylation as demonstrated by sensitivity to protein phosphatase treatment. The applicability and the potential of this system for the analysis of the various mammalian PKC isoenzymes is discussed.

Sequence of a rat cDNA encoding the ERK1-MAP kinase.

Mitogen-activated protein (MAP) kinases are cytoplasmic and/or nuclear protein kinases which are activated by one or several signal transduction pathways from the cell surface into the nucleus. Their activity is regulated by phosphorylation on Tyr as well as on Ser/Thr residues. A cDNA encoding the rat ERK1 member of the MAP kinase family was isolated and sequenced. The longest cDNA consisted of 1875 nucleotides and coded for a polypeptide of 380 amino acids with a predicted M(r) of 42987.

Downregulation of protein kinase C-gamma is independent of a functional kinase domain.

Prolonged activation of protein kinase C (PKC) types alpha and beta by tumor-promoting phorbol esters leads to desensitization of the phorbol ester response, downregulation of protein kinase C activity and depletion of the protein kinase C polypeptide. When the gamma isoenzyme of PKC is transiently expressed in COS-1 cells and exposed to phorbol esters, PKC-Gamma is downregulated in COS cells although these cells do not normally express this subtype. A point mutation in the putative ATP-binding site (Lys-380----Met-380) of the protein kinase C gamma isoenzyme which results in a kinase-deficient enzyme does not interfere with this downregulation. Our results suggest that autophosphorylation or constitutive signalling through the protein kinase C-gamma kinase domain is not a prerequisite for downregulation of PKC activity.

Immunocharacterization of delta- and zeta-isoenzymes of protein kinase C in rat renal mesangial cells.

The isoforms of protein kinase C (PKC) present in rat mesangial cells were identified by immunoblot analysis with antibody raised against isotype-specific peptides. In addition to the previously observed alpha- and epsilon-subspecies, mesangial cells also express the delta- and zeta-isoenzymes of PKC. On exposure to phorbol 12,13-dibutyrate (PDB) a complete depletion of PKC-delta is observed within 8 h. Removal of PDB results in a recovery of PKC-delta. In contrast, PKC-zeta is unaffected by addition or removal of PDB.

Stimulation of MAP kinase by v-raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau.

A proportion of the microtubule-associated protein, tau, is in an elevated state of phosphorylation in foetal and adult brain whereas all of the tau in paired helical filaments, which are characteristic of Alzheimer's disease is hyperphosphorylated; it is important therefore to elucidate the mechanisms that regulate tau phosphorylation. Here we describe results that show that although MAP kinase can hyperphosphorylate tau in vitro, activation of MAP kinase in transformed fibroblasts does not result in hyperphosphorylation of transfected tau, whereas glycogen synthase kinase-3 beta (GSK-3 beta) when co-transfected with tau does result in tau hyperphosphorylation. The findings imply that GSK-3 beta may be a stronger candidate than MAP kinase for inducing tau hyperphosphorylation in vivo.

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cdelta by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation.

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCdelta with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.

Arrangement and expression of integrated adenovirus type 12 DNA in the transformed hamster cell line HA12/7: amplification of Ad12 and c-myc DNAs and evidence for hybrid viral-cellular transcripts.

In the genome of the adenovirus type 12 (Ad12)-transformed hamster cell line HA12/7 about three copies of the viral DNA are fixed by integration. The results of blot-hybridization, molecular cloning, and nucleotide sequencing experiments suggest a model for the arrangement of Ad12 DNA molecules in which the left hand terminus of one of the Ad12 DNA copies is linked to unique hamster DNA. The right hand end of this DNA molecule is fused to an inverted copy of a left terminal approximately 4.3 kb fragment of Ad12 DNA. This ensemble is followed by the second Ad12 DNA copy whose right terminus is again joined to an inverted, supernumerary left terminal approximately 4.3 kb Ad12 DNA fragment. There is a third Ad12 DNA copy whose right terminus is linked to cellular DNA. In this sequence arrangement, the left terminus of Ad12 DNA is overrepresented, as had been shown earlier (S. Stabel, W. Doerfler and R.R. Friis (1980) J. Virol. 36, 22-40). In the presented model, cellular DNA sequences are interspersed in between the three copies of Ad12 DNA. In the left terminus of the integrated Ad12 DNA, transcription of RNA is initiated which extends out into cellular DNA. The interviral DNA junctions are also transcribed. The c-myc gene in cell line HA12/7 is amplified about 10-fold and considerably more c-myc RNA has been identified in the Ad12-transformed cells than in BHK21 or in LSH hamster cells. It has been shown previously that the E1 region of Ad12 DNA is transcribed into mRNA in HA12/7 cells (Ortin et al. (1976) J. Virol. 20, 355-372). It remains to be investigated whether c-myc amplification and expression are related to the transformed phenotype of HA12/7 cells.

Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.

Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, and eta have been expressed using the baculovirus expression system. The partially purified PKC subtypes have been studied for their substrate specificities and phospholipid-independent activation by various chemically different nontumor- and tumor-promoting agents, as well as their inhibition of kinase activity by staurosporine and two related compounds. An endogenous PKC-like kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition. Protamine sulfate was most efficiently phosphorylated by all of the PKC subtypes tested, although this phosphorylation was independent of phosphatidylserine (PS) and diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Except for PKC-zeta, all subtypes tested phosphorylated myelin basic protein (MBP), histone, or a peptide derived from the pseudosubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varying extents. Among the various agents tested, TPA most efficiently stimulated the kinase activities of the PKC subtypes in a phospholipid-dependent manner. Phorbol 12,13-dibutyrate (PDBu) was less effective than TPA but displayed no major difference among the subtypes. Activation of PKC-alpha by bryostatin-1 reached only half of the TPA response whereas the other subtypes were activated more effectively. The weak tumor promoter resiniferonol 9,13,14-orthophenyl acetate (ROPA) mainly stimulated PKC-alpha and PKC-gamma at 1 microM concentration, whereas PKC-epsilon and PKC-eta were much less activated. Sapintoxin D, mezerein, indolactam V, and resiniferatoxin at concentrations of 1-100 nM preferentially activated PKC-alpha in a DAG-like manner, whereas at 1 microM other subtypes were activated as well. Preferential activation of PKC-alpha was also noted for tinyatoxin and thapsigargin, but their mode of activation is unclear because these two compounds did not compete for the phorbol ester binding of the PKC subtypes as the other agents did. Of the three PKC inhibitors tested, staurosporine most efficiently inhibited kinase activity of the PKC subtypes, whereas K252a and CGP 41251 were at least 10 times less effective. However, K252a showed certain specificity for inhibition of PKC-alpha, and CGP 41251 failed to inhibit PKC-epsilon and PKC-zeta. Given the different substrate specificities and modes of activation by various tumor-promoting and nontumor-promoting agents, as well as the different sensitivities towards different inhibitors, our results indicate a divergence of individual PKC subtypes in signal transduction.

Protein kinase C--a family affair.

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.