Role of the phosphatase PP4 in the activation of JNK-1 in prostate carcinoma cell lines PC-3 and LNCaP resulting in increased AP-1 and EGR-1 activity

The specific signaling connections between the mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK-1) and phosphatases PP4 and M3/6, affecting the family of early nuclear factors, is complex and remains poorly understood. JNK-1 regulates cellular differentiation, apoptosis and stress responsiveness by up-regulating early nuclear factors such as c-Jun, a member of the activating protein (AP-1) family, and the Early Growth Factor (EGR-1). C-Jun, when phosphorylated by c-Jun N-terminal kinase (JNK-1) associates with c-Fos to form the AP-1 transcription factor that activates gene expression. We have investigated the regulation of the JNK-1 kinase by co-transfecting phosphatases PP4 and M3/6 in prostate cancer cell lines PC-3 and LNCaP, which have been previously stimulated with human EGF or cisplatin. Co-transfections of plasmids expressing the JNK-1 and the serine/threonine phosphatases PP4 resulted in a significant increase in JNK-1 activity in both PC3 and LNCaP cells. In contrast, co-transfection of JNK-1 with the dual specific phosphatase serine/threonine M3/6 showed only a marginal effect in JNK-1 activity. The phosphatase M3/6 also failed in blocking the induction of JNK-1 activity observed in presence of PP4. The higher activity of JNK-1 was associated with increased activities of the factors c-Jun/AP-1 and EGR-1. This suggests that JNK-1 activity in PC-3 and LNCaP cells requires not only active PP4 for stable maintenance but also suggests that the relative degree of phosphorylation of multiple cellular components is the determinant of JNK-1 stability.

Efeitos de inibidores da biossíntese do ergosterol, de proteínas quinases e do citoesqueleto em Trypanosoma cruzi / Effect of sterol biosynthesis inhibitors, of kinases proteins and the cytoskeleton in Trypanosoma cruzi

Trypanosoma cruzi é o agente etiológico da doença de Chagas. Novos compostos vêm sendo desenvolvidos tendo como alvo a biossíntese e função de esteróis, já que T. cruzi requer esteróis endógenos específicos para o crescimento e sobrevivência. Inibidores da biossíntese do ergosterol (SBIs) são drogas comumente usadas contra doenças fúngicas... Ambas as drogas inibiram a multiplicação dos parasitos, com IC50/72 h de 24,3 and 4,5 mM, respectivamente. O segundo grupo de drogas estudadas neste trabalho foram WSP 413, WSP 414, WSP 415, WSP 488, WSP 501 e WSP 561, inibidores específicos da D24(25)-esterol metiltransferase. Todas as drogas inibiram a multiplicação dos parasitos a concentrações muito baixas, com valores de IC50/72 h de 0,53, 0,59, 0,53, 0,48, 0,44 e 0,48 mM, respectivamente. Os dois inibidores da SQS e WSP 488, WSP 501 e WSP 561 induziram mudanças morfológicas drásticas nos parasitos incluindo (a) destacamento de um dos folhetos da membrana, formando bolhas, (b) destacamento da membrana do corpo celular e do flagelo dos microtúbulos subpeliculares e axonemais, respectivamente, (c) aumento da bolsa flagelar, (d) aparecimento de um vacúolo localizado próximo à bolsa flagelar, que parece corresponder ao vacúolo contrátil, (e) inchaço da mitocôndria, com o aparecimento de estruturas concêntricas formadas pelas invaginações da membrana mitocondrial interna, (f) alterações no núcleo de algumas células, onde a cromatina aparece em grumos, como descrito para células apoptóticas, e (g) bloqueio do processo de divisão celular... WSP 414 e BPQ-OH foram testados em células infectadas com amastigotas intracelulares de T. cruzi. WSP 414 a 1 mM praticamente eliminou a infecção das células, e os parasitos sobreviventes não se diferenciaram em tripomastigotas. BPQ-OH teve pouco efeito sobre a infecção. Ambas as drogas causaram alterações morfológicas nos parasitos sem afetar a célula hospedeira. O terceiro grupo de drogas testadas neste trabalho foram inibidores de proteínas quinases. Staurosporina, genisteína e wortmanina inibiram o crescimento dos parasitos... O quarto e último grupo de drogas testadas foram os inibidores de polimerização e despolimerização de actina e microtúbulos. Foram testadas três drogas: jasplakinolida, citocalasina D e nocodazol. Todas as três drogas inibiram o crescimento dos parasitos e a divisão celular, mas tiveram poucos efeitos sobre a ultraestrutura dos parasitos.

Enhanced expression of a calcium-dependent protein kinase from the moss Funaria hygrometrica under nutritional starvation.

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss Funaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA of Funaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in the Funaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene from Funaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of the Funaria gene shows extensive homology with CDPKs from higher plants, 73% identity with the Fragaria CDPK and 71% identity with CDPK isoform 7 of Arabidopsis. Phylogenetic analysis revealed that the Funaria CDPK is closer to the CDPKs from higher plants like strawberry and Arabidopsis as compared to those from lower plants such as the liverwort Marchantia, the green alga Chlamydomonas or another moss Tortula. Northern analysis shows enhanced expression of the CDPK transcript within 24-48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.