RESUMO
The Kinetworks trade mark multi-immunoblotting technique was used to evaluate the expressions of 78 protein kinases, 24 protein phosphatases and phosphorylation states of 31 phosphoproteins in thoracic spinal cord tissue from control subjects and patients having the sporadic form of amyotrophic lateral sclerosis (ALS). In both the cytosolic (C) and particulate (P) fractions of spinal cord from ALS patients as compared with controls, there were increased levels of calcium/calmodulin-dependent protein kinase kinase (CaMKK; C = 120% increase/P = 580% increase;% change, compared with control), extracellular regulated kinase 2 (ERK2; C = 120% increase/P = 170% increase), G protein-coupled receptor kinase 2 (GRK2; C = 140% increase/P = 140% increase), phospho-Y279/216 glycogen synthase kinase 3 alpha/beta (GSK3alpha/beta; C = 90% increase/P = 220% increase), protein kinase B alpha (PKBalpha; C = 360% increase/P = 200% increase), phospho-T638 PKCalpha/beta (C = 630% increase/P = 170% increase), cGMP-dependent protein kinase (PKG; C = 100% increase/P = 75% increase), phospho-T451 dsRNA-dependent protein kinase (PKR; C = 2600% increase/P = 3330% increase), ribosomal S6 kinase 1 (RSK1; C = 750% increase/P = 630% increase), phospho-T389 p70 S6 kinase (S6K; C = 1000% increase/P = 460% increase), and protein-tyrosine phosphatase 1 delta (PTP1delta; C = 43% increase/P = 70% increase). Cytosolic increases in phospho-alpha-S724/gamma-S662 adducin (C = 15650% increase), PKCalpha (C = 100% increase) and PKCzeta (C = 190% increase) were found in ALS patients as compared with controls, while particulate increases in cAMP-dependent protein kinase (PKA; 43% increase), protein kinase C beta (PKCbeta; 330% increase), and stress-activated protein kinase beta (SAPKbeta; 34% increase) were also observed. Cyclin-dependent kinase-associated phosphatase (KAP) was apparently translocated, as it was reduced (31% decrease) in cytosolic fractions but elevated (100% increase) in particulate fractions of ALS spinal cord tissue. Our observations indicate that ALS is associated with the elevated expression and/or activation of many protein kinases, including PKCalpha, PKCbeta, PKCzeta and GSK3alpha/beta, which may augment neural death in ALS, and CaMKK, PKBalpha, Rsk1, S6K, and SAPK, which may be a response to neuronal injury that potentially can mitigate cell death.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Fosfoproteínas Fosfatases/biossíntese , Proteínas Quinases/biossíntese , Medula Espinal/enzimologia , Idoso , Esclerose Lateral Amiotrófica/patologia , Animais , Estabilidade Enzimática , Congelamento , Quinase 3 da Glicogênio Sintase/análise , Quinase 3 da Glicogênio Sintase/biossíntese , Humanos , Isoenzimas/análise , Isoenzimas/biossíntese , Fosfoproteínas Fosfatases/análise , Mudanças Depois da Morte , Proteína Quinase C/análise , Proteína Quinase C/biossíntese , Proteínas Quinases/análise , Proteína Fosfatase 1 , Ratos , Ratos Sprague-Dawley , Medula Espinal/química , Medula Espinal/patologia , Fatores de TempoRESUMO
The factors responsible for ALS-parkinsonism dementia complex (ALS-PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin-linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non-water soluble compounds from washed cycad flour and have identified three sterol beta-d-glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non-toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT-mediated biotin-dUTP nick-end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase-3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS-PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.
Assuntos
Esclerose Lateral Amiotrófica/etiologia , Colesterol/análogos & derivados , Neurônios/efeitos dos fármacos , Fitosteróis/isolamento & purificação , Fitosteróis/toxicidade , Sementes/química , Esclerose Lateral Amiotrófica/complicações , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Bioensaio , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Colesterol/química , Cycas , Demência/complicações , Demência/etiologia , Glucose/análogos & derivados , Glucose/química , Glucosídeos/isolamento & purificação , Glucosídeos/toxicidade , Guam , Humanos , Técnicas In Vitro , Masculino , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Neurotoxinas/isolamento & purificação , Neurotoxinas/toxicidade , Transtornos Parkinsonianos/complicações , Transtornos Parkinsonianos/etiologia , Técnicas de Patch-Clamp , Fitosteróis/química , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Sitosteroides/isolamento & purificação , Sitosteroides/toxicidade , Estigmasterol/análogos & derivados , Estigmasterol/química , Estigmasterol/isolamento & purificação , Estigmasterol/toxicidadeRESUMO
OBJECTIVE: The aim of this study was to study the pattern of protein kinase expression in a culture model of epithelial ovarian carcinogenesis. METHODS: Cultures of normal human ovarian surface epithelium (OSE), OSE from women with BRCA1 mutations, a cell culture model of preneoplastic (SV40 T-antigen-immortalized, nontumorigenic) and neoplastic (SV40-E-cadherin transfected, tumorigenic) OSE, and three ovarian cancer cell lines were used to represent OSE phenotypes of different genetic backgrounds and at different, progressive stages of neoplastic transformation. The protein kinase network signaling was studied by Western blotting, simultaneously using multiple antibodies for specific protein kinases. RESULTS: High levels of cGMP-dependent protein kinase were found in normal and preneoplastic OSE, but were absent in neoplastic OSE. In contrast, expression of MEK6 was detected exclusively in neoplastic OSE. The expressions of casein kinase II (CK2), p38 mitogen-activated protein kinase (MAPK), cyclin-dependent kinase, and the phosphatidylinositol 3-kinase (PI3K) effectors Akt2 and p70 S6 kinase (S6K) were several-fold higher in neoplastic OSE than in normal OSE, whereas the expressions of the MAPKs extracellular signal-regulated kinases ERK1 and -2 were unchanged. Importantly, constitutive phosphorylation of Akt2 and p70 S6K, as found in neoplastic OSE, was also observed in overtly normal OSE from women with predisposing BRCA1 gene mutations. CONCLUSIONS: These data demonstrate that different repertoires of downstream signaling proteins, particularly those of the MEK6-p38 MAPK-CK2 pathway and the PI3K pathway, are correlated with phenotypic manifestations of a cell culture model of OSE at progressive stages in the development of ovarian cancer. Changes in PI3K effectors are already found in overtly normal OSE from women with BRCA1 mutations.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Ovarianas/enzimologia , Lesões Pré-Cancerosas/enzimologia , Proteínas Quinases/biossíntese , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/fisiologia , Western Blotting , Caderinas/genética , Progressão da Doença , Eletroforese em Gel de Poliacrilamida , Epitélio/enzimologia , Epitélio/patologia , Feminino , Genes BRCA1/genética , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Ovário/enzimologia , Ovário/patologia , Fosforilação , Lesões Pré-Cancerosas/patologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Propriedades de Superfície , Transfecção , Células Tumorais CultivadasRESUMO
Excessive activation of N-methyl-D-aspartate (NMDA) receptors leads to cell death in human embryonic kidney-293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA-mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA-mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down-regulation. The region of NR2A that provides the PKC-induced enhancement of cell death was localized to a discrete segment of the C-terminus. Use of isoform-specific PKC inhibitors showed that Ca(2+)- and lipid-dependent PKC isoforms (cPKCs), specifically PKCbeta1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by an in vitro kinase assay was also increased in NR1A/NR2A-transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C-terminus of NR2A to accentuate cell death in NR1/NR2A-transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.
Assuntos
Morte Celular/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , Proteína Quinase C/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Fracionamento Celular , Linhagem Celular , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Immunoblotting , Indóis/farmacologia , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Estrutura Terciária de Proteína , Subunidades Proteicas , Pirróis/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/genética , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Heat shock proteins (hsp) have important roles in the regulation and protection of both prokaryotic and eukaryotic cells, especially during environmental stress. Hsps are also important bacterial virulence factors. We investigated whether bacterial hsp60 can alter epithelial cell mitogen-activated protein kinase (MAPK) signaling and cell proliferation. Human skin keratinocytes (HaCaT cell line) were cultured in the presence of hsp60 purified from Actinobacillus actinomycetemcomitans, an important oral pathogen. Protein kinases in the ERK1/2 and p38 MAPK signaling pathways were probed with kinase-specific and phosphorylation-site-specific antibodies on Western blots. In quiescent cultures, hsp60 increased ERK1/2 phosphorylation in a sustained manner and p38 phosphorylation transiently. Hsp60 also increased epithelial cell proliferation by about 30%. Inhibition of the ERK1/2 pathway by PD 98059 (a MEK1 inhibitor) reversed partially ERK1/2 phosphorylation and totally cell proliferation indicating that the ERK1/2 MAPK pathway is involved in the hsp60-induced cell growth. This was supported by findings that hsp60 stimulated phosphorylation of RSK1/2 and cyclic AMP response element-binding protein and increased expression of transcription factors c-Jun and c-Fos. Recombinant human hsp60 did not alter ERK1/2 or p38 phosphorylation and had no effect on epithelial cell proliferation. Inhibition of p38 MAPK pathway by SB 203580 increased both ERK1/2 phosphorylation and cell proliferation demonstrating that the inhibitor can either directly or indirectly activate the ERK1/2 MAPK pathway. The results show that exogenous bacterial hsp60 is able to activate ERK1/2 phosphorylation and thereby cause increased epithelial proliferation. In case of mucosal infection this effect may either lead to increased wound repair or participate in the pathological mechanism of some bacterial diseases that involve increased epithelial proliferation.
Assuntos
Infecções Bacterianas/metabolismo , Infecções Bacterianas/fisiopatologia , Divisão Celular/fisiologia , Chaperonina 60/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa , Actinobacillus/metabolismo , Actinobacillus/patogenicidade , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/metabolismo , Chaperonina 60/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , MAP Quinase Quinase Quinases/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Since autocrine regulation of HGF-Met is implicated in many forms of human cancer, we investigated whether the predisposition to develop ovarian cancer in women with hereditary ovarian cancer syndromes involves changes in the expression of HGF-Met by the tissue of origin of epithelial ovarian cancers, the ovarian surface epithelium (OSE). We compared cultures of normal OSE from women with (FH-OSE) (n=20) and with no (NFH-OSE) (n=48) family histories of ovarian cancer, SV40 Tag immortalized OSE lines (IOSE, n=5) and ovarian cancer cell lines (n=3). Cultures derived from 21/22 women with NFH-OSE and 13/13 women with FH-OSE expressed Met mRNA initially. After two to three passages, Met was downregulated in 37% of NFH-OSE cultures but persisted in 100% of FH-OSE cultures and ovarian cancer lines, like other epithelial differentiation markers that are stabilized in FH-OSE and neoplasia. HGF and Met mRNA were concomitantly expressed by NFH-OSE from only three of 32 women but in FH-OSE from eight of 13 women, and also in five of five IOSE and two of three ovarian cancer lines. Conditioned media from FH-OSE, but not NFH-OSE, contained immunoreactive HGF and induced cohort migration which was inhibited by neutralizing HGF antibody. Several signaling molecules of the PI3K pathway, including Akt2 and p70 S6K, were constitutively activated in FH-OSE from six of six women but in NFH-OSE from only four of eight women. Exogenous HGF was mitogenic in OSE, and that effect was regulated through the MAP kinase (ERK1/ERK2) and FRAP/p70 S6K pathways. The proliferative response to HGF was greater in NFH-OSE than in FH-OSE cultures. The results show that FH-OSE cultures differ from NFH-OSE by increased stability of Met expression and by HGF secretion. Constitutive phosphorylation of kinases and a diminished growth response to HGF suggest the presence of autocrine regulation in FH-OSE. In analogy with other cell types where an autocrine HGF-Met loop has been implicated in tumorigenic transformation, this change in FH-OSE may play a role in the enhanced susceptibility to ovarian carcinogenesis in women with hereditary ovarian cancer syndromes.
Assuntos
Células Epiteliais/citologia , Fator de Crescimento de Hepatócito/biossíntese , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-met/biossíntese , Comunicação Autócrina , Movimento Celular , Transformação Celular Neoplásica , Feminino , Predisposição Genética para Doença , Humanos , MorfogêneseRESUMO
A murine model of motoneuron disease, the pmn/pmn mouse, shows a reduction in the retrograde transport of fluorescent probes applied directly onto the cut end of sciatic nerve. Brain-derived neurotrophic factor (BDNF), when co-applied with fluorescent tracers, increases the number of retrograde labelled motoneurons. We demonstrate here that spinal cord tissue from pmn/pmn mice had significantly reduced phosphatidylinositol 3-kinase activity and expression in the particulate fraction compared to controls, without changes in the activities or expression of the downstream kinases, protein kinase B/Akt or Erk1. Systemic administration of BDNF augmented phosphatidylinositol 3-kinase specific activity in spinal cord tissue from pmn/pmn and control mice, with a greater elevation in the particulate fractions of pmn/pmn mice than in controls. We examined the effect of inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase on the retrograde labelling of motoneurons, 24h following the direct application of inhibitors and Fluorogold to the cut end of sciatic nerve in control and pmn/pmn mice (labelling index). The mitogen-activated protein kinase kinase inhibitor PD 98059 had no effect on the labelling index in control or pmn/pmn mice. In the absence of exogenous BDNF, phosphatidylinositol 3-kinase inhibitors reduced the number of labelled motoneurons in control mice, without changing the labelling index in pmn/pmn. Co-application of phosphatidylinositol 3-kinase inhibitors with BDNF to the cut end of sciatic nerve blocked the action of BDNF on retrograde labelling in pmn/pmn mice. These results indicate that the retrograde labelling of motoneurons is mediated by phosphatidylinositol 3-kinase-dependent and -independent pathways. In pmn/pmn mice, phosphatidylinositol 3-kinase activity in spinal neurons is below the level required for optimal retrograde labelling of motoneurons and labelling can be augmented by the administration of growth factors stimulating phosphatidylinositol 3-kinase activity. The data indicate that phosphatidylinositol 3-kinase activity is important in the uptake and/or retrograde transport of substances by motoneurons and is altered in this model of motoneuron diseases.
Assuntos
Doença dos Neurônios Motores/enzimologia , Neurônios Motores/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Camundongos , Camundongos Mutantes , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas/metabolismo , Medula Espinal/enzimologiaRESUMO
The role of protein kinases in the inhibition of TNF-alpha associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-alpha and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-alpha produced only a modest and delayed activation of Akt. In the presence of TNF-alpha, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-alpha itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 microM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-alpha. Furthermore, the inhibition of the Mek1/Mek2-->ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-alpha-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Pirofosfato de Cálcio/farmacologia , Caspases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Apoptose/imunologia , Caspase 3 , Inibidores de Caspase , Caspases/biossíntese , Caspases/metabolismo , Cristalização , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/imunologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/imunologia , Repressão Enzimática/efeitos dos fármacos , Repressão Enzimática/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/biossíntese , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
A p38(MAPK) homolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38-alpha isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G(2)/M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.
Assuntos
Fertilização , Isoenzimas/metabolismo , Meiose , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/fisiologia , Estrelas-do-Mar/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Fase G2 , Humanos , Isoenzimas/química , Microinjeções , Proteínas Quinases Ativadas por Mitógeno/química , Dados de Sequência Molecular , Fosforilação , Homologia de Sequência de Aminoácidos , Estrelas-do-Mar/fisiologia , Tirosina/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2 in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.
Assuntos
Anisomicina/farmacologia , Arsenitos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Caseína Quinase II , Ativação Enzimática , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Arginine vasopressin (AVP) and lysophosphatidic acid (LPA) have been shown to stimulate protein kinase C (PKC) and mitogen-activated protein (MAP) kinases and the proliferation of vascular smooth muscle cells. However, the actions of these two agents in cardiomyocytes are less well understood. To investigate the signal transduction pathways of AVP and LPA, freshly isolated adult rat cardiomyocytes were examined. Both AVP and LPA induced concentration- and time-dependent stimulation of the phosphotransferase activities of p90 ribosomal S6 kinases (RSK) and their upstream activators, extracellularly regulated kinases (ERK) 1 and 2. The activation of ERK1 and ERK2 by LPA was PKC- and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent. However, AVP-induced activation of RSK2, a downstream substrate of ERK1 and ERK2, was PKC-dependent and PI 3-kinase-independent. AVP and LPA were also observed to increase the phosphotransferase activity of p70 ribosomal protein S6 kinase (p70 S6K) in a time- and concentration-dependent manner. The activation of p70 S6K by LPA and AVP was PI 3-kinase-dependent. PKC was necessary in AVP- but not in LPA-induced activation of p70 S6K. Since RSK and p70 S6K have been implicated in the regulation of translational control of protein synthesis, we concluded that AVP and LPA may stimulate the growth of cardiomyocytes through these two protein kinase cascades.
Assuntos
Arginina Vasopressina/farmacologia , Coração/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Miocárdio/enzimologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Técnicas In Vitro , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peso Molecular , Miocárdio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/efeitos dos fármacosRESUMO
Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.
Assuntos
Precondicionamento Isquêmico Miocárdico , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Isquemia Miocárdica/enzimologia , Miocárdio/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Anisomicina/farmacologia , Circulação Coronária , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase 4 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Isquemia Miocárdica/patologia , Inibidores da Síntese de Proteínas/farmacologia , Coelhos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 microM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 microM, 1 min), but had no effect on PMA-treated cells (0.5 microM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46+/-9 and 54+/-3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.
Assuntos
Pirofosfato de Cálcio/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Sesquiterpenos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Carcinógenos/farmacologia , Cicloexanos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Neutrófilos/enzimologia , O-(Cloroacetilcarbamoil)fumagilol , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
The myelin basic protein (MBP)-phosphorylating enzymes present during maturation and early embryogenesis of the sea star (Pisaster ochraceus) were investigated. The major maturation-activated MBP kinase (p45 Mapk) was molecularly cloned based on tryptic sequence information obtained with the purified enzyme and shown to be highly related to human Erk1 with 76% amino acid identity. Kinase assays and immunoblotting studies revealed that Mapk remained highly active until 12 h post-fertilization (PF), after which it declined. By 4 days PF, Mapk protein was no longer detectable. At 3 h PF, about half of the detectable MBP phosphotransferase activity could be attributed to a 75 kDa protein kinase that was distinct from Mapk. Like Mapk, this protein phosphorylated MBP mostly on threonine residues, but it failed to phosphorylate a peptide (APRTPGGRR) based upon the Thr-97 MAP kinase phosphorylation site in MBP. Rather, it phosphorylated a peptide (AAQKRPSQRTKYLA) patterned after the N-terminus of MBP. Our studies also showed a dramatic increase in MBP phosphotransferase activity occurred by 4 days PF that arose from a third kinase that phosphorylated MBP solely on serine residues. This kinase exhibited the following substrate substrate preference: AAQKRPSQRTKYLA, peptide substrate for S6 kinases (AKRRRLSSLRASTSKSESSQK) > MBP > histone H1 > prota-mine > casein > APRTPGGRR. This kinase was not appreciably affected by addition of phosphatidylserine/diacylglycerol, or the staurosporine analogue Roche Compound 3, but it was partly inhibited by a protein kinase C pseudosubstrate peptide. Gel filtration analysis revealed an apparent molecular mass of 41 kDa for the enzyme. Therefore, at least two novel MBP-phosphorylating enzymes distinct from Mapk are preferentially activated following fertilization and early embryogenesis of the sea star.
Assuntos
Fertilização , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Básica da Mielina/metabolismo , Fosfotransferases/fisiologia , Proteínas Quinases/fisiologia , Estrelas-do-Mar/enzimologia , Sequência de Aminoácidos , Animais , Ciclo Celular , Células Cultivadas , Cromatografia em Gel , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Oócitos/enzimologia , Oócitos/fisiologia , Testes de Precipitina , Proteína Quinase C/metabolismo , Homologia de Sequência de Aminoácidos , Estrelas-do-Mar/embriologia , Fatores de TempoRESUMO
Two ribosomal protein S6 kinases (i.e., pp52(S6K) and pp70(S6K)) of the p70 S6 kinase family were markedly activated during meiotic maturation of Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52(S6K) by approximately 50,000-fold with a specific enzyme activity of 1.6 micromol per min per mg. The purified enzyme apparently featured the N- and C-terminal regions of pp70(S6K) as it immunoreacted with antibodies directed to peptides patterned after these amino acid sequences in mammalian pp70(S6K). pp52(S6K) was inhibited by fluoride (IC(50) approximately 60 mM), but was relatively insensitive to beta-glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn(2+), Zn(2+), and Ca(2+). The consensus sequence for substrate phosphorylation was determined to be RXXSXR, which was partially distinct from mammalian p70(S6K) in its requirement for an amino-terminal arginine. Phosphorylation of ribosomal protein S6 by p52(S6K) occurred exclusively on serine on at least five tryptic peptides. Inhibition of sea star p52(S6K) phosphotransferase activity after treatment with protein serine/threonine phosphatases confirmed that p52(S6K) was still regulated by phosphorylation. The sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein-serine phosphatase 2A and the heat shock protein 60. The association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A-specific antibodies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases S6 Ribossômicas/química , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Citosol/metabolismo , Immunoblotting , Íons , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfoproteínas Fosfatases/farmacologia , Fosforilação , Ligação Proteica , Proteína Fosfatase 2 , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Análise de Sequência , Coloração pela Prata , Estrelas-do-Mar/embriologia , Estrelas-do-Mar/enzimologia , Especificidade por SubstratoRESUMO
The effects of tail-vein insulin injection (2 U/kg) on the regulation of protein-serine kinases in hindlimb skeletal muscle were investigated in hyperinsulinemic hypertensive fructose-fed (FF) animals that had been fasted overnight. Basal protein kinase B (PKB) activity was elevated about twofold in FF rats and was not further stimulated by insulin. Phosphatidylinositol 3-kinase (PI3K), which lies upstream of PKB, was increased approximately 3.5-fold within 2-5 min by insulin in control rats. Basal and insulin-activated PI3K activities were further enhanced up to 2-fold and 1.3-fold, respectively, in FF rats. The 70-kDa S6 kinase (S6K) was stimulated about twofold by insulin in control rats. Both basal and insulin-stimulated S6K activity was further enhanced up to 1.5-fold and 3.5-fold, respectively, in FF rats. In control rats, insulin caused a 40-50% reduction of the phosphotransferase activity of the beta-isoform of glycogen synthase kinase 3 (GSK-3beta), which is a PKB target in vitro. Basal GSK-3beta activity was decreased by approximately 40% in FF rats and remained unchanged after insulin treatment. In summary, 1) the PI3K --> PKB --> S6K pathway was upregulated under basal conditions, and 2) insulin stimulation of PI3K and S6K activities was enhanced, but both PKB and GSK-3 were refractory to the effects of insulin in FF rats.
Assuntos
Frutose , Hipertensão/induzido quimicamente , Hipertensão/enzimologia , Insulina/fisiologia , Músculo Esquelético/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática/fisiologia , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Hiperinsulinismo/enzimologia , Hipertensão/metabolismo , Proteína Básica da Mielina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/metabolismoRESUMO
BACKGROUND: Abnormalities of the cyclin-dependent protein kinase (CDK) machinery have been linked to cancer development. Hyperphosphorylation of the retinoblastoma (Rb) protein results in release from inhibition of progression through the G1 phase of the cell cycle. Hyperexpression of CDK1 and CDK2 may enable progression through late G1, S and the G2 phases of the cell cycle. METHODS/RESULTS: To investigate tumor-associated protein kinase activities, control and tumor samples were fractionated by MonoS chromatography and tested for their ability to phosphorylate histone H1. Two major peaks of histone H1 phosphotransferase activity were resolved. The first appeared in the flow through fractions, and occasionally showed enhanced activity in the tumor samples, whilst the second was consistently increased and eluted at approximately 0.4 M NaCl. Western immunoblotting with CDK1 and PSTAIRE antibodies confirmed the co-elution of CDK1 and CDK2 with the second peak. Next, the phosphotransferase activities (following specific immunoprecipitation) and protein levels of CDK1, 2, 4, and 6 were determined in human colon cancer samples and their respective controls. CDK4 activity was elevated in only 3 of 7 tumor samples (range 40-160%) relative to control samples from the same patients, whereas a significant increase in CDK6 activity was observed in the same group (p < 0.05). This contrasted sharply with the universal activations of CDK1 (up to 18-fold, p < 0.01, n = 12) and CDK2 (up to 17-fold, p < 0.05) in the same groups. CONCLUSIONS: CDK1 especially, and to a lesser extent CDK2 and CDK6 demonstrate the most consistent biochemical activation in human colon cancer and may represent targets for pharmacological intervention. Cellular proliferation as gauged by MIB1 was not directly correlated with the amplitude of activation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Neoplasias do Colo/enzimologia , Quinases Ciclina-Dependentes/biossíntese , Quinases Ciclina-Dependentes/metabolismo , Antígenos Nucleares , Biomarcadores Tumorais/metabolismo , Western Blotting , Proteína Quinase CDC2/biossíntese , Proteína Quinase CDC2/metabolismo , Fracionamento Químico , Cromatografia por Troca Iônica , Quinase 2 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Testes de Precipitina , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteína do Retinoblastoma/metabolismoRESUMO
A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A's important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases (PAK1 and PAK3) in the PP2A complex, indicating these kinases may associate with PP2A. The existence of protein kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the kinases revealed that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258-1261) and casein kinase IIalpha (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997) Science 276, 952-955), the present data provide compelling evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Glutationa Transferase/metabolismo , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 2 , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Quinases Ativadas por p21RESUMO
Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.
Assuntos
Precondicionamento Isquêmico Miocárdico , Isquemia Miocárdica/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Caseína Quinase II , Cromatografia por Troca Iônica , Ativação Enzimática , Feminino , Immunoblotting , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Infarto do Miocárdio/enzimologia , Coelhos , Proteínas Quinases S6 Ribossômicas/metabolismoRESUMO
The control of glucose uptake and glycogen metabolism by insulin in target organs is in part mediated through the regulation of protein-serine/threonine kinases. In this study, the expression and phosphotransferase activity levels of some of these kinases in rat heart ventricle were measured to investigate whether they might mediate the shift in the energy dependency of the developing heart from glycogen to fatty acids. Following tail-vein injection of overnight fasted adult rats with 2 U of insulin per kg body weight, protein kinase B (PKB), the 70-kDa ribosomal S6 kinase (S6K), and casein kinase 2 (CK2) were activated (30-600%), whereas the MAP/extracellular regulated kinases (ERK)1 and ERK2 were not stimulated under these conditions. When the expression levels of the insulin-activated kinases were probed with specific antibodies in ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats, phosphatidylinositol 3-kinase (PI3K), PKB, S6K, and CK2 were downregulated (40-60%) with age. By contrast, ventricular glycogen synthase kinase-3beta (GSK3beta) protein levels were maintained during postnatal development. Similar findings were obtained when the expression of these kinases was investigated in freshly isolated ventricular myocytes, where they were detected predominantly in the cytosolic fraction of the myocytes. Compared to other adult rat tissues such as brain and liver, the levels of PI3K, PKB, S6K, and GSK3beta were relatively low in the heart. Even though CK2 protein and activity levels were reduced by approximately 60% in 365 day as compared to 1-day-old rats, expression of CK2 in the adult heart was as high as detected in any of the other rat tissues. The high basal activities of CK2 in early neonatal heart may be associated with the proliferating state of myocytes.