RESUMO
Spontaneous and glucocorticoid (fluocinolone acetonide, FA)-induced apoptosis of primary mouse thymocytes was inhibited by protein kinase C (PKC) activators such as bryostatin-1 and phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) within the first 2-4 h of incubation but was enhanced upon prolonged treatment. Only the anti-apoptotic but not the pro-apoptotic effect of TPA was completely suppressed by the PKC inhibitor Goe 6983 and moderately inhibited by Goe 6976. Immunoblot analysis revealed distinct PKC alpha, beta, delta, eta, theta, mu and zeta signals, a very faint PKCepsilon and no PKCgamma signal. Upon prolonged TPA treatment all PKC isoenzymes became downregulated, albeit at different rates (PKCdelta>alpha>mu>beta,theta>>eta,zeta). No significant generation of caspase-derived catalytic PKC fragments, as found to be produced upon induction of apoptosis and to be pro-apoptotic in other systems, was observed in FA- or TPA-treated thymocytes. It is concluded that the early anti-apoptotic effect of TPA depends on the activation of n-type PKC isoenzymes, whereas stimulation of spontaneous and FA-induced apoptosis by TPA ensues, at least partially, from a downregulation (or inactivation) of anti-apoptotic PKC species, i.e. in primary thymocytes PKC activation is primarily involved in a negative regulation of apoptosis.
Assuntos
Apoptose , Glucocorticoides/farmacologia , Proteína Quinase C/metabolismo , Timo/efeitos dos fármacos , Animais , Briostatinas , Carbazóis/farmacologia , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Feminino , Fluocinolona Acetonida/antagonistas & inibidores , Fluocinolona Acetonida/farmacologia , Immunoblotting , Indóis/farmacologia , Isoenzimas/metabolismo , Lactonas/farmacologia , Macrolídeos , Camundongos , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Timo/citologiaRESUMO
A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase Cdelta (PKCdelta(cat)) cDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns. DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKCdelta(cat) and PKCdelta with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKCdelta(cat) or PKCdelta and DIK expression constructs.
Assuntos
Isoenzimas/metabolismo , Queratinócitos/enzimologia , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Domínio Catalítico , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 21/genética , Éxons/genética , Perfilação da Expressão Gênica , Humanos , Íntrons/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Proteína Quinase C-delta , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Estaurosporina/farmacologia , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Using isoenzyme-specific antibodies, we have performed an immunoblot analysis of the PKC isoenzyme pattern during the course of TPA-induced tumor promotion in the epidermis of NMRI mice. The TPA-sensitive PKC isoforms alpha, delta, straightepsilon, eta, nu (and TPA-insensitive PKCzeta), but not PKCbeta and gamma, were found to be expressed in both normal and neoplastic epidermis. The immune signals of all TPA-sensitive PKC isoforms were moderately and reversibly attenuated upon a single TPA treatment. Using different antibodies against PKCeta and PKCmu, this apparent downregulation could mainly be attributed to epitope changes of these enzymes, whereas for the other PKC species no such conclusion could be drawn. Except for PKCstraightepsilon, no substantial long-term attenuation of the immune signals of the other PKC isoforms occurred upon chronic phorbol ester treatment (i.e., 14 applications of 5 nmol TPA each over 7 weeks), which led to tumor development in initiated mouse skin. Specific PKC activity (related to tissue weight) was 40-50% lower in TPA-treated as compared with control epidermis whereby no clearcut difference was found between single and chronic TPA treatment. Benign and malignant skin tumors generated according to the initiation-promotion protocol did not exhibit consistent alterations in the immune pattern of the PKC isoenzymes with the exception of a decrease of PKCstraightepsilon and an increase of PKCmicro signal in carcinomas. Our data indicate that, in contrast with earlier assumptions, no general long-lasting PKC downregulation plays a critical role in skin tumor promotion.
Assuntos
Proteína Quinase C/metabolismo , Neoplasias Cutâneas/enzimologia , Pele/enzimologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Feminino , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos , Pele/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/induzido quimicamente , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
The heterogeneous nuclear ribonucleoprotein K protein recruits a diversity of molecular partners and may act as a docking platform involved in such processes as transcription, RNA processing, and translation. We show that K protein is tyrosine-phosphorylated in vitro by Src and Lck. Treatment with H(2)O(2)/Na(3)VO(4), which induces oxidative stress, stimulated tyrosine phosphorylation of K protein in cultured cells and in intact livers. Tyrosine phosphorylation increased binding of Lck and the proto-oncoprotein Vav to K protein in vitro. Oxidative stress increased the association of K protein with Lck and Vav, suggesting that tyrosine phosphorylation regulates the ability of K protein to recruit these effectors in vivo. Translation-based assay showed that K protein is constitutively bound to many mRNAs in vivo. Native immunoprecipitated K protein-mRNA complexes were disrupted by tyrosine phosphorylation, suggesting that the in vivo binding of K protein to mRNA may be responsive to the extracellular signals that activate tyrosine kinases. This study shows that tyrosine phosphorylation of K protein regulates K protein-protein and K protein-RNA interactions. These data are consistent with a model in which functional interaction of K protein is responsive to changes in the extracellular environment. Acting as a docking platform, K protein may bridge signal transduction pathways to sites of nucleic acid-dependent process such as transcription, RNA processing, and translation.
Assuntos
RNA/metabolismo , Ribonucleoproteínas/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Transdução de Sinais , Tirosina , Quinases da Família src/metabolismoRESUMO
The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein recruits a diversity of molecular partners that are involved in signal transduction, transcription, RNA processing, and translation. K protein is phosphorylated in vivo and in vitro by inducible kinase(s) and contains several potential sites for protein kinase C (PKC) phosphorylation. In this study we show that K protein is phosphorylated in vitro by PKCdelta and by other PKCs. Deletion analysis and site-directed mutagenesis revealed that Ser302 is a major K protein site phosphorylated by PKCdelta in vitro. This residue is located in the middle of a short amino acid fragment that divides the two clusters of SH3-binding domains. Mutation of Ser302 decreased the level of phosphorylation of exogenously expressed K protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser302 is also a site for PKC-mediated phosphorylation in vivo. In vitro, PKCdelta binds K protein via the highly interactive KI domain, an interaction that is blocked by poly(C) RNA. Mutation of Ser302 did not alter the K protein-PKCdelta interaction in vitro, suggesting that phosphorylation of this residue alone is not sufficient to alter this interaction. Instead, binding of PKCdelta to K protein in vitro and in vivo was greatly increased by K protein phosphorylation on tyrosine residues. The ability of PKCdelta to bind and phosphorylate K protein may serve not only to alter the activity of K protein itself, but K protein may also bridge PKCdelta to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKCdelta-K protein interaction may serve to meet cellular needs at sites of active transcription, RNA processing and translation in response to changing extracellular environment.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , RNA Nuclear Heterogêneo/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Células Cultivadas , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Ribonucleoproteínas Nucleares Heterogêneas , Camundongos , Mutação , Fosforilação , Proteína Quinase C-delta , RNA/genética , RNA Nuclear Heterogêneo/genética , Ribonucleoproteínas/genética , Serina/genética , Serina/fisiologiaRESUMO
Recently, we reported that, in contrast to protein kinase C (PKC)alpha and betaII, PKCdelta does not require phosphorylation of a specific threonine (Thr505) in the activation loop for catalytic competence (Stempka et al. (1997) J. Biol. Chem. 272, 6805-6811). Here, we show that the acidic residue glutamic acid 500 (Glu500) in the activation loop is important for the catalytic function of PKCdelta. A Glu500 to valine mutant shows 76 and 73% reduced kinase activity toward autophosphorylation and substrate phosphorylation, respectively. With regard to thermal stability and inhibition by the inhibitors Gö6976 and Gö6983 the mutant does not differ from the wild type, indicating that the general conformation of the molecule is not altered by the site-directed mutagenesis. Thus, Glu500 in the activation loop of PKCdelta might take over at least part of the role of the phosphate groups on Thr497 and Thr500 of PKCalpha and betaII, respectively. Accordingly, PKCdelta exhibits kinase activity and is able to autophosphorylate probably without posttranslational modification. Autophosphorylation of PKCdelta in vitro occurs on Ser643, as demonstrated by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides of autophosphorylated PKCdelta wild type and mutants. A peptide containing this site is phosphorylated also in vivo, i.e. in recombinant PKCdelta purified from baculovirus-infected insect cells. A Ser643 to alanine mutation indicates that autophosphorylation of Ser643 is not essential for the kinase activity of PKCdelta. Probably additional (auto)phosphorylation site(s) exist that have not yet been identified.
Assuntos
Ácido Glutâmico/química , Isoenzimas/química , Proteína Quinase C/química , Serina/química , Animais , Catálise , Estabilidade Enzimática/genética , Isoenzimas/genética , Cinética , Mutagênese Sítio-Dirigida , Mutação/genética , Fosfopeptídeos/análise , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C-delta , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes alpha, betaI, and betaII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative "PKC kinase" for permissive activation. Expression of PKCalpha in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCalpha and PKCbetaII could be identified as Thr497 and Thr500, respectively. We report here that PKCdelta, contrary to PKCalpha, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCdelta expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCalpha and PKCbetaII, respectively, is not essential for obtaining a catalytically competent conformation of PKCdelta. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCdelta and that at least this PKC isoenzyme differs from the isotypes alpha, betaI, and betaII regarding the permissive activation by a PKC kinase.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Alanina/química , Animais , Baculoviridae , Inibidores Enzimáticos/farmacologia , Escherichia coli , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Proteína Quinase C-delta , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Spodoptera , Relação Estrutura-Atividade , Treonina/químicaRESUMO
A new category of epitopes of human thyroglobulin is being described defined by a monoclonal antibody (TuTg 1). The epitope shows a very variable expression in normal individuals as well as in different thyroid diseases. According to gel filtration data the epitope is located at the intact molecule and is very sensitive to tryptic digestion. The monoclonal antibody was used in an IRMA system for plasma measurements in the follow-up of thyroid cancer patients. In individuals with high epitope expression the sensitivity to detect recurrency during T4 treatment was higher than a sensitive commercial TG IRMA.
Assuntos
Anticorpos Monoclonais/imunologia , Tireoglobulina/imunologia , Neoplasias da Glândula Tireoide/metabolismo , Cromatografia em Gel , Epitopos , Humanos , Tireoglobulina/biossíntese , Neoplasias da Glândula Tireoide/imunologiaRESUMO
It has become evident in recent years that autoimmune thyroglobulin (Tg) antibodies of Graves disease and Hashimoto's thyroiditis show a restricted epitope repertoire compared to Tg heteroantibodies. We have produced monoclonal antibodies (Mab) against human Tg by the hybridoma technique and the epitope specificity was determined by crossblocking experiments. Six noncrossreactive Mabs were used in a double determinant IRMA system for plasma Tg measurements. Sensitivity of the assays was between 1 and 2 ng/ml, intraassay variation less than 5%. Recovery experiments with added Tg were performed in 25 Graves sera with elevated Tg autoantibodies. Monoclonal antibody Tg13 showed an unusual strong interference with autoantibodies resulting in a very low recovery in all sera (median: less than 10%). In further studies Tg was digested by trypsin and after Western blotting, the resulting fragments were incubated with different Mab antibodies, a polyclonal antibody and 10 different Graves sera with high Tg autoantibodies. In contrast to all other mabs only Mab Tg13 showed several low molecular weight bands between 17 and 50 KD. The major bands recognized by Mab Tg13 corresponded to bands obtained by the autoimmune sera, which showed a very homogeneous band pattern. We conclude that Mab Tg13 is specific for an autoimmunodominant B cell epitope of human Tg.
Assuntos
Anticorpos Monoclonais/imunologia , Doenças Autoimunes/imunologia , Epitopos/imunologia , Genes Dominantes/imunologia , Tireoglobulina/imunologia , Anticorpos Monoclonais/análise , Doenças Autoimunes/sangue , Doenças Autoimunes/genética , Western Blotting , Epitopos/genética , Genes Dominantes/genética , Doença de Graves/sangue , Doença de Graves/genética , Doença de Graves/imunologia , Humanos , Hibridomas/imunologia , Peso Molecular , Tireoglobulina/análise , Tireoglobulina/genética , Tireoidite Autoimune/sangue , Tireoidite Autoimune/genética , Tireoidite Autoimune/imunologiaRESUMO
The expression of six antigenic epitopes on plasma Tg and tissue-derived Tg was investigated using mouse mabs in immunoradiometric assays. The sensitivity of the assays ranged between 0.5 and 1.5 micrograms l-1. Plasma thyroglobulin of 15 normal individuals and 18 patients with metastatic, differentiated thyroid cancer was analysed. Whereas five monoclonals produced values comparable to the conventional RIA system, the monoclonal antibody Tg 158 did not detect any Tg in the normal individuals and only low levels in two of the cancer patients with the highest Tg levels. In contrast, dilutions of extracts of three different euthyroid goitre samples and three different samples of differentiated thyroid cancer revealed no difference in the antigenic expression of all six epitopes, including the Tg 158 epitope. Papain digestion of purified Tg resulted in a 76 kD fragment which showed immunoreactivity for Tg 158 as well as for Tg 11. No interference of the binding of Tg 158 by T3, T4 or DIT could be detected. We conclude that the monoclonal antibody Tg 158 detects an epitope which is present in thyroid tissue-derived Tg but not in circulating thyroglobulin. This antibody might be useful for the investigation of the secretory process of thyroglobulin from the thyrocytes into the circulation.