RESUMEN
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.
Asunto(s)
Isoenzimas/metabolismo , Queratinocitos/enzimología , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Dominio Catalítico , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 21/genética , Exones/genética , Perfilación de la Expresión Génica , Humanos , Intrones/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C-delta , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Estaurosporina/farmacología , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Ácido Glutámico/química , Isoenzimas/química , Proteína Quinasa C/química , Serina/química , Animales , Catálisis , Estabilidad de Enzimas/genética , Isoenzimas/genética , Cinética , Mutagénesis Sitio-Dirigida , Mutación/genética , Fosfopéptidos/análisis , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C-delta , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteína Quinasa C/metabolismo , Brioestatinas , Caspasa 3 , Sistema Libre de Células , Cicloheximida/farmacología , Citarabina/farmacología , Fructosa-Bifosfato Aldolasa/metabolismo , Humanos , Lactonas/farmacología , Macrólidos , Oligopéptidos/farmacología , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología , Células U937RESUMEN
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.
Asunto(s)
Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Alanina/química , Animales , Baculoviridae , Inhibidores Enzimáticos/farmacología , Escherichia coli , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C-delta , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Spodoptera , Relación Estructura-Actividad , Treonina/químicaRESUMEN
Rottlerin, a compound from Mallotus philippinensis, is shown to inhibit protein kinases with some specificity for PKC. To some extent, the novel inhibitor is able to differentiate between PKC isoenzymes, with IC50 values for PKC delta of 3-6 microM, PKC alpha,beta,gamma of 30-42 microM and PKC epsilon,eta,zeta of 80-100 microM. Inhibition of PKC appears, at least in part, to be due to a competition between rottlerin and ATP. Among the protein kinases tested, only CaM-kinase III is suppressed by rottlerin as effectively as PKC delta. The chemical structure of rottlerin might serve as a basis for the development of novel inhibitors with improved selectivity for a distinct PKC isoenzyme, such as PKC delta, or for CaM-kinase III.
Asunto(s)
Acetofenonas/farmacología , Benzopiranos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Unión Competitiva , Creatina Quinasa/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Técnicas In Vitro , Proteína Oncogénica pp60(v-src)/antagonistas & inhibidores , Proteínas Recombinantes , PorcinosRESUMEN
An antiserum raised against an epsilon PKC-specific peptide recognizes epsilon PKC with an apparent molecular weight of 97 kDa in cytosol of mouse brain. No cross-reaction with alpha, beta, gamma PKC or the delta PKC-like p76-kinase is observed. Epsilon PKC is mainly present in brain. Just traces of this PKC isoenzyme can be detected in some other murine tissues. Ontogenetic studies indicate that the amount of epsilon PKC in murine brain increases constantly and reaches a maximal level at day 7 after birth. Upon TPA activation epsilon PKC is translocated from the cytosol to the particulate fraction in a brain homogenate.
Asunto(s)
Proteína Quinasa C/inmunología , Factores de Edad , Animales , Transporte Biológico , Western Blotting , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Compartimento Celular/efectos de los fármacos , Citosol/enzimología , Femenino , Isoenzimas/clasificación , Isoenzimas/inmunología , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos , Proteína Quinasa C/clasificación , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Distribución TisularRESUMEN
We isolated and sequenced mouse lipocortin I cDNA clones from a lambda gt10 cDNA library prepared from Swiss 3T3 mRNA. The homology with human lipocortin I at the amino acid level is 86%. When confluent layers of Swiss 3T3 cells were stimulated with 10% fetal calf serum, expression of lipocortin I was strongly stimulated. In parallel, DNA synthesis was induced with a peak at 24 hours after glucocorticoid treatment indicating induction of cell proliferation. In the absence of serum glucocorticoid treatment provoked neither induction of DNA synthesis nor expression of lipocortin I. We conclude that serum contains an unidentified factor, which acts synergistically with glucocorticoids on cell proliferation and lipocortin I expression.
Asunto(s)
Clonación Molecular , ADN/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Glucocorticoides/farmacología , Glicoproteínas/genética , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Secuencia de Aminoácidos , Animales , Anexinas , Secuencia de Bases , División Celular/efectos de los fármacos , Línea Celular , ADN/aislamiento & purificación , Fluocinolona Acetonida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Indometacina/farmacología , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , ARN Mensajero/genética , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
Hyperplasiogenic and tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate induce the sequential transient expression of the proto-oncogenes c-fos and c-myc and the ornithine decarboxylase gene in mouse skin in vivo. This sequence of biochemical events probably depends on an activation of protein kinase C by these agents. The non-irritant skin mitogens 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and ethyl phenyl propiolate do not increase the expression of these genes to a comparable extent. Thus, 12-O-tetradecanoylphorbol-13-acetate and 12-O-retinoylphorbol-13-acetate induce epidermal hyperproliferation by different biochemical mechanisms as do 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and ethylphenylpropiolate.
Asunto(s)
Ornitina Descarboxilasa/genética , Ésteres del Forbol/toxicidad , Proto-Oncogenes , Piel/efectos de los fármacos , Animales , Femenino , Hiperplasia , Ratones , ARN Mensajero/análisis , Piel/metabolismo , Piel/patología , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
The expression of ornithine decarboxylase (ODC) mRNA after treatment of murine Swiss 3T3 cells with the tumor promoter TPA was studied. The induction of ODC mRNA was detectable after 20-40 min, peaked after 60-120 min and declined within 24 hrs. Using an in vitro nuclear transcription assay, we found that the polymerase II density on the ODC gene is not affected by TPA treatment. Additionally, we were able to detect stable ODC mRNAs in cycloheximide pretreated fibroblasts. These two different experimental approaches lead us to the interpretation that in Swiss 3T3 cells TPA controls ODC expression predominantly at the post transcriptional level by prolonging the half-life of ODC-mRNA.
Asunto(s)
Ornitina Descarboxilasa/genética , Acetato de Tetradecanoilforbol/farmacología , Animales , Cicloheximida/farmacología , Dactinomicina/farmacología , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Ratones , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacosRESUMEN
(1) The induction of vitellogenin synthesis in chicken liver by an estrogen implant as well as the decline of vitellogenin synthesis and nuclear receptor concentration after withdrawal of the implant were studied. For the detection of vitellogenin the SAC immunoprecipitation technique was used. The nuclear receptor decreases very rapidly and somewhat earlier than the capacity of the liver to synthesize vitellogenin. (2) The inhibition by antiestrogens of the estrogen-induced vitellogenin synthesis as well as of the accumulation of the estrogen-receptor complex in the nucleus was investigated. Tamoxifen as well as the recently described 1,1,2,2-tetramethyl-1,2-bis(4'-hydroxyphenyl)ethane were found to be true antiestrogens in the chicken liver, i.e. they inhibit estrogen-induced vitellogenin synthesis but themselves cause no induction.