Modificações pós-traducionais durante o processo de amastigogênese do Trypanosoma cruzi / Post-translational modifications during the amastigogenesis of Trypanosoma cruzi

A doença de Chagas é uma doença negligenciada causada pelo protozoário Trypanosoma cruzi constituindo-se em um problema de saúde pública em vários países da América Latina. No seu complexo ciclo de vida, o protozoário passa por quatro estágios diferentes: tripomastigota metacíclica, amastigota, tripomastigota sanguíneo e epimastigota, que permitem sua sobrevivência nos diferentes ambientes com os quais o parasita entra em contato. A diferenciação dos tripomastigotas de T. cruzi em amastigotas (amastigogênese) ocorre com grandes mudanças morfológicas, estruturais e metabólicas no parasita e pode ser reproduzido in vitro por exemplo, pela acidificação do meio extracelular. Apesar dos vários trabalhos descritos na literatura, o processo ainda não é totalmente compreendido. A participação de NO na transdução de sinal durante a amastigogênese, sugerida por dados não publicados de nosso grupo, assim como a via de sinalização dependente de AMPc, foram o foco do presente estudo. A indução da amastigogênese foi obtida por incubação de tripomastigotas em meio de cultura acidificado (pH 6,0) e os parâmetros estudados comparados com parasitas controle (meio de cultura, pH 7,4). Estudamos a variação no perfil de nucleotídios cíclicos (AMPc, GMPc), de quinases (PKA, MAPK- ERK1/2), de uma fosfatase (PP2A), assim como o perfil de proteínas fosforiladas, S-nitrosiladas e nitradas até 6 h do início da amastigogênese. O processo foi dividido nas etapas: inicial (até 60 minutos) e tardio (em torno de 3-4 h), caracterizados por um aumento de formas amastigotas na etapa tardia. Houve um aumento de aproximadamente 17 vezes no nível de AMPc nos primeiros 15 minutos da amastigogênese (meio pH 6,0), seguido por aumento discreto no nível de PKA fosforilada, utilizado como indicador de atividade enzimática, este mais evidente na etapa tardia (360 minutos). Quanto à subunidade catalítica fosforilada da MAPK (ativa), há uma aparente diminuição no nível de fosforilação na fase inicial (30 minutos) e aumento na etapa tardia (120 minutos) do processo de amastigogênese. Quanto ao perfil geral de fosforilação de proteínas, há uma diminuição de fosforilação em torno de 30 minutos, seguida de aumento de fosforilação em proteínas de aproximadamente 5 e 100 kDa, mas de maneira geral, não se observaram grandes mudanças nesse perfil com a metodologia utilizada. Quanto às modificações por NO e seus derivados, foram observadas modificações por S-nitrosilação e nitração das proteínas, além do aumento de GMPc em torno de 60 minutos. Embora essas modificações modulem a atividade biológica de uma grande diversidade de proteínas, seu papel biológico não foi explorado.8 Em resumo, nossos resultados apontam para uma variação no perfil de fosforilação, S-nitrosilação e nitração de proteínas, além do aumento de AMPc e GMPc ao longo do processo de amastigogênese in vitro, com a via de sinalização dependente de quinases/ fosfatases e de óxido nítrico ocorrendo ao longo do processo de amastigogênese

Chagas disease is a neglected disease caused by the parasite Trypanosoma cruzi and is a public health problem in several Latin American countries. In its complex life cycle, the protozoan goes through four different stages: metacyclic trypomastigote, amastigote, blood trypomastigote and epimastigote, which allow its survival in the different environments which the parasite comes into contact. The differentiation of T. cruzi trypomastigotes into amastigotes (amastigogenesis) occurs with large morphological, structural and metabolic changes in the parasite and can be reproduced in vitro by, for example, acidification of the extracellular medium. Despite the many data described in the literature, the process is not yet fully understood. The participation of NO in signal transduction during amastigogenesis, suggested by unpublished data from our group, as well as the cAMP-dependent signaling pathway, were the focus of the present study. The induction of amastigogenesis was obtained by incubating trypomastigotes in acidified culture medium (pH 6.0) and the studied parameters compared with control parasites (culture medium, pH 7.4). We studied the variation in the profile of cyclic nucleotides (cAMP, cGMP), kinases (PKA, MAPK-ERK1 / 2), phosphatase (PP2A), as well as the profile of phosphorylated, S-nitrosylated and nitrated proteins up to 6 h. onset of amastigogenesis. The process was divided into early (up to 60 minutes) and late (around 3-4 hours), characterized by an increase in amastigote forms in the late stage. There was an approximately 17-fold increase in cAMP level in the first 15 minutes of amastigogenesis (pH 6.0 medium), followed by a slight increase in phosphorylated PKA level, most evident in the late stage (360 minutes). As for the phosphorylated catalytic subunit of MAPK (active), there is an apparent decrease in the phosphorylation level in the early phase (30 minutes) and increase in the late stage (120 minutes) of the amastigogenesis process. As for the general protein phosphorylation profile, there is a decrease in phosphorylation around 30 minutes, followed by an increase in phosphorylation of proteins (approximately 5 and 100 kDa), but overall, no major changes were observed in this profile with the methodology used. As for modifications by NO and its derivatives, modifications were observed by S-nitrosylation and protein nitration, besides the increase of cGMP around 60 minutes. Although these modifications modulate the biological activity of a wide range of proteins, their biological role has not been explored. In summary, our results point to a variation in phosphorylation, S-nitrosylation and nitration profile of proteins, as well as an increase in cAMP and cGMP along the amastigogenesis process, implicating kinases / phosphatases and nitric oxide dependent signaling pathways in this differentiation

TPL2 expression is correlated with distant metastasis and poor prognosis in colorectal cancer.

Tumor progression locus 2 (TPL2) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K). The present study aimed to elucidate the clinicopathological significance and prognostic role of TPL2 expression in colorectal cancer (CRC) through immunohistochemistry. In the present study, the correlations between TPL2 expression and clinicopathological parameters, including survival rate, were investigated using 262 archival paraffin-embedded CRC tissue samples. In addition, the correlation between TPL2 expression and tumor-infiltrating lymphocytes was evaluated using immunoscore. High TPL2 expression was found in 40.1% of the 262 CRCs analyzed. Patients with high TPL2 expression had frequent distant metastasis compared to patients with low TPL2 expression. However, there was no significant correlation between high TPL2 expression and other clinicopathological parameters. High TPL2 expression was significantly correlated with low immunoscore. In subgroup analysis based on distant metastasis, there was a significant correlation between high TPL2 expression and low immunoscore in only CRCs without distant metastasis. High TPL2 expression significantly correlated with poor overall survival. In both CRCs with and without distant metastasis, CRCs with high TPL2 expression showed worse prognosis compared to CRCs with low TPL2 expression (P = .034 and P = .010, respectively). Taken together, our results showed that high TPL2 expression was significantly correlated with distant metastasis and low immunoscore. In addition, TPL2 expression can be useful for predicting the prognosis of patients with CRC.

Healing potential of zerumbone ointment on experimental full-thickness excision cutaneous wounds in rat.

Zerumbone is a monocyclic sesquiterpene compound. Based on report, it is the predominant bioactive compound from the rhizomes of Zingiber zerumbet. The study was undertaken to evaluate the therapeutic effects of topical zerumbone on excision wounds in rats. A 1% (w/w) simple ointment containing zerumbone was applied topically (100 mg ointment per rat) once a day on full-thickness excision wounds created on rats. The wound tissue was removed and used for estimation of antioxidant activity and to observe histopathological changes. Immunohistochemical staining was performed to study the expression pattern of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß1 and collagen IV. Zerumbone exhibited antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans and Candida tropicali. Zerumbone ointment has potent wound healing capacity as evident from the wound contraction on 15th post wounding day. The histopathological examinations of healed wound sections showed greater tissue regeneration, more fibroblasts and angiogenesis in zerumbone ointment-treated group. VEGF, TGF-ß1 and collagen IV expression were also correlative with the healing pattern observed. Zerumbone possesses potent antioxidant activity by increasing superoxide dismutase, catalase, glutathione and decreased lipid peroxidation. The synergistic effects of both antimicrobial and antioxidant activities in zerumbone are deduced to have accelerated the wound repair. The results demonstrate that zerumbone possessed strong wound healing potential and can be exploited to accelerate excision wound healing.

Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development.

Arterial occlusive diseases are major causes of morbidity and mortality. Blood flow to the affected tissue must be restored quickly if viability and function are to be preserved. We report that disruption of the mixed-lineage protein kinase (MLK) - cJun NH2-terminal kinase (JNK) signaling pathway in endothelial cells causes severe blockade of blood flow and failure to recover in the murine femoral artery ligation model of hindlimb ischemia. We show that the MLK-JNK pathway is required for the formation of native collateral arteries that can restore circulation following arterial occlusion. Disruption of the MLK-JNK pathway causes decreased Dll4/Notch signaling, excessive sprouting angiogenesis, and defects in developmental vascular morphogenesis. Our analysis demonstrates that the MLK-JNK signaling pathway is a key regulatory mechanism that protects against ischemia in arterial occlusive disease.

Expression of TAK1/TAB1 expression in non-small cell lung carcinoma and adjacent normal tissues and their clinical significance.

The purpose of this study was to investigate the expression of transforming growth factor beta-activated kinase 1 (TAK1) and its activation ligand, TAK1-binding protein 1 (TAB1), in non-small cell lung carcinoma (NSCLC) and adjacent normal tissues and to analyze the relevance between TAK1 and TAB1 protein expression and the pathological features of NSCLC patients. Surgical resection NSCLC specimens were collected from 74 patients undergoing surgery in our hospital from September 2003 to July 2008; tumor-adjacent normal tissue specimens were collected as controls. All cases were pathologically confirmed after surgery, and pathological data were complete for all patients. The expression of TAK1/TAB1 proteins in NSCLC and adjacent cancer tissues was detected by immunohistochemical analysis. The correlation between TAK1/TAB1 protein expression and the clinicopathological features and outcome of NSCLC was assessed. The positive expression ratio of TAK1 in NSCLC tissue was 63.5%, which was significantly higher than that in tumor-adjacent normal tissue (31.1%). The positive expression ratio of TAB1 in NSCLC tissue was 51.4%, which was significantly higher than that in tumor-adjacent normal tissue (24.3%). Further analysis showed that positive protein expression of TAK1 and TAB1 was unrelated to patient gender, age, tumor size, degree of differentiation, and history of smoking (P>0.05) but was significantly related to clinical stage and lymph node metastasis (P<0.05). Additionally, the expression of TAK1 as well as TAB1 was negatively related to NSCLC patient prognosis, and patients with positive protein expression had a significantly lower 5-year survival rate than those with negative protein expression (P<0.05). TAK1/TAB1 expression in NSCLC tissue is significantly increased and closely associated with patient clinical prognosis. These two proteins are likely to become new therapeutic targets for the treatment of NSCLC.

Suppression of Tak1 promotes prostate tumorigenesis.

More than 30% of primary prostate cancers contain a consensus deletion of an approximately 800 kb locus on chromosome 6q15.1. The MAP3K7 gene, which encodes TGF-ß activated kinase-1 (Tak1), is a putative prostate tumor suppressor gene within this region whose precise function remains obscure. In this study, we investigated the role of Tak1 in human and murine prostate cancers. In 50 well-characterized human cancer specimens, we found that Tak1 expression was progressively lost with increasing Gleason grade, both within each cancer and across all cancers. In murine prostate stem cells and Tak1-deficient prostatic epithelial cells, Tak1 loss increased proliferation, migration, and invasion. When prostate stem cells attenuated for Tak1 were engrafted with fetal urogenital mesenchyme, the histopathology of the grafts reflected the natural history of prostate cancer leading from prostatic intraepithelial neoplasia to invasive carcinoma. In the grafts containing Tak1-suppressed prostate stem cells, p38 and c-jun-NH(2)-kinase activity was attenuated and proliferation was increased. Together, our findings functionally validate the proposed tumor suppressor role of Tak1 in prostate cancer.

Assessment of the pathway of apoptosis involving PAR-4, DAXX and ZIPK proteins in CLL patients and its relationship with the principal prognostic factors.

Par-4 (prostate apoptosis response-4) protein was originally found upregulated in prostate tumor cells undergoing apoptosis. Then it was further identified as a proapoptotic protein upregulated both in normal and leukemic lymphocytes. The aim of our study was to assess PAR-4 protein expression in the B cells of CLL patients and to examine its relationship with the expression of other proteins involved in the apoptosis process, such as DAXX, ZIPK and BCL-2. We found a positive relationship between PAR-4 and BCL-2 protein expression. Additionally, there was a positive correlation between PAR-4 and both DAXX and ZIPK protein expression. The results of our research were also analyzed in association with the principal CLL prognostic factors. There was a positive correlation between the expression of PAR-4 protein and the lactate dehydrogenase (LDH) serum concentration (p < 0.005). The expression of PAR-4 protein in B cells correlated positively with the percentage of CD38(+) cells (p < 0.05), as well as with CD38(+)/ZAP-70(+) cells (p < 0.05). Moreover, we found a close relationship between LPL protein expression or LPL/ADAM29 MFI ratio and PAR-4 protein expression. Our results confirm the significance of apoptosis deregulation in CLL, and suggest a possible relationship between PAR-4 expression and the clinical course of the disease. This however requires further investigation.

Pancreatic adenocarcinoma upregulated factor promotes metastasis by regulating TLR/CXCR4 activation.

Pancreatic adenocarcinoma upregulated factor (PAUF) is overproduced in certain types of cancer. However, little is known of the tumorigenic function of PAUF. In this study, we report the X-ray crystal structure of PAUF and reveal that PAUF is a mammalian lectin normally found in plant lectins. We also identify PAUF as an endogenous ligand of Toll-like receptor 2 (TLR2) and TLR4 by screening extracellular domain receptor pools. We further confirmed the specificity of the PAUF-TLR2 interaction. PAUF induces extracellular signal-regulated kinase (ERK) phosphorylation and activates the IKK-ß-mediated TPL2/MEK/ERK signaling pathway through TLR2. In agreement with the result of TLR2-mediated ERK activation by PAUF, PAUF induces increased expression of the protumorigenic cytokines RANTES and MIF in THP-1 cells. However, PAUF does not fully activate Iκ-B-α signaling pathways in THP-1 cells, and fails to translocate the p65 subunit of the nuclear factor-κB (NF-κB) complex into the nucleus, resulting in no NF-κB activation. Surprisingly, we found that PAUF also associated with the CXC chemokine receptor (CXCR4)-TLR2 complex and inhibited CXCR4-dependent, TLR2-mediated NF-κB activation. Together, these findings suggest that the new cancer-associated ligand, PAUF, may activate TLR-mediated ERK signaling to produce the protumorigenic cytokines, but inhibits TLR-mediated NF-κB signaling, thereby facilitating tumor growth and escape from innate immune surveillance.

[Regulation of the JNK signaling pathway by dual leucine zipper kinase DLK.].

The C-Jun NH2-terminal kinase (JNK) belongs to the evolutionarily conserved sub-group of mitogen-activated protein (MAP) kinases family. Many studies have shown that JNK pathway plays physiological roles in cell proliferation, differentiation, migration and apoptosis, and its deregulation has been associated with developmental defects and various human diseases. Dual leucine zipper kinase (DLK) is a member of the mixed-lineage kinases that performs important cellu-lar functions as a MAP triple kinase (MAPKKK) in regulating the JNK signaling pathway. In this paper, we described the DLK protein structures, physiological roles, and their functional interactions with JNK signaling, as well as the molecular mechanisms underlying their involvement in various human diseases.

Short double-stranded RNAs of specific sequence activate ribosomal TAK1-D and induce a global inhibition of translation.

We have previously shown that short double-stranded RNAs of specific sequence induce phosphorylation in the activation loop of splicing variant D of the transforming growth factor beta-activated protein kinase 1 (TAK1-D). Here, we further characterize this novel function of TAK1-D and the mechanisms of this dsRNA-triggered phenomenon. Using a dominant negative TAK1-D mutant we demonstrate that TAK1-D activation is functionally required to trigger the activation of p38 MAP kinase and c-JUN terminal kinase and to induce cell death in NCI-H460 cells. While total TAK1-D protein was found in the cytoplasm as well as in the ribosomal fraction, activated TAK1-D phosphorylated on T184 and T187 in the activation loop was found to be exclusively associated with the 80S ribosome. The association of TAK1-D with the ribosome suggests an involvement in translation-dependent signaling and we demonstrate here that dsRNA-mediated activation of TAK1-D leads to a downregulation of mRNA translation. In addition, we show that TAK1-D is also phosphorylated after the induction of ribotoxic stress. Our data indicate that TAK1-D plays a role in the signaling events triggered by selected types of ribotoxic stress.

Activation of the dual-leucine-zipper-bearing kinase and induction of beta-cell apoptosis by the immunosuppressive drug cyclosporin A.

Post-transplant diabetes is an untoward effect often observed under immunosuppressive therapy with cyclosporin A. Besides the development of peripheral insulin resistance and a decrease in insulin gene transcription, a beta-cell toxic effect has been described. However, its molecular mechanism remains unknown. In the present study, the effect of cyclosporin A and the dual leucine-zipper-bearing kinase (DLK) on beta-cell survival was investigated. Cyclosporin A decreased the viability of the insulin-producing pancreatic islet cell line HIT in a time- and concentration-dependent manner. Upon exposure to the immunosuppressant fragmentation of DNA, the activation of the effector caspase-3 and a decrease of full-length caspase-3 and Bcl(XL) were observed in HIT cells and in primary mature murine islets, respectively. Cyclosporin A and tacrolimus, both potent inhibitors of the calcium/calmodulin-dependent phosphatase calcineurin, stimulated the enzymatic activity of cellular DLK in an in vitro kinase assay. Immunocytochemistry revealed that the overexpression of DLK but not its kinase-dead mutant induced apoptosis and enhanced cyclosporin A-induced apoptosis to a higher extent than the drug alone. Moreover, in the presence of DLK, the effective concentration for cyclosporin A-caused apoptosis was similar to its known IC(50) value for the inhibition of calcineurin activity in beta cells. These data suggest that cyclosporin A through inhibition of calcineurin activates DLK, thereby leading to beta-cell apoptosis. This action may thus be a novel mechanism through which cyclosporin A precipitates post-transplant diabetes.

Deletion of a small consensus region at 6q15, including the MAP3K7 gene, is significantly associated with high-grade prostate cancers.

PURPOSE: Chromosome 6q14-21 is commonly deleted in prostate cancers, occurring in approximately 22% of all tumors and approximately 40% of metastatic tumors. However, candidate prostate tumor suppressor genes in this region have not been identified, in part due to the large and broad nature of the deleted region implicated in previous studies. EXPERIMENTAL DESIGN: We first used high-resolution Affymetrix single nucleotide polymorphism arrays to examine DNA from malignant and matched nonmalignant cells from 55 prostate cancer patients. We identified a small consensus region on 6q14-21 and evaluated the deletion status within the region among additional 40 tumors and normal pairs using quantitative PCR and fluorescence in situ hybridization. We finally tested the association between the deletion and Gleason score using the Fisher's exact test. RESULTS: Tumors with small, interstitial deletions at 6q14-21 defined an 817-kb consensus region that is affected in 20 of 21 tumors. The MAP3K7 gene is one of five genes located in this region. In total, MAP3K7 was deleted in 32% of 95 tumors. Importantly, deletion of MAP3K7 was highly associated with higher-grade disease, occurring in 61% of tumors with Gleason score >or=8 compared with only 22% of tumors with Gleason score

The protein kinase genes MAP3K epsilon 1 and MAP3K epsilon 2 are required for pollen viability in Arabidopsis thaliana.

We have used reverse-genetic analysis to investigate the function of MAP3K epsilon 1 and MAP3K epsilon 2, a pair of closely related Arabidopsis thaliana genes that encode protein kinases. Plants homozygous for either map3k epsilon 1 or map3k epsilon 2 displayed no apparent mutant phenotype, whereas the double-mutant combination caused pollen lethality. Transmission of the double-mutant combination through the female gametophyte was normal. Tetrad analysis performed using the Arabidopsis quartet mutation demonstrated that the pollen-lethal phenotype segregated at meiosis with the map3k epsilon 1;map3k epsilon 2 genotype. We used transmission electron microscopy to determine that double-mutant pollen grains develop plasma membrane irregularities following pollen mitosis I. Analysis of the subcellular localization of a yellow fluorescent protein (YFP):MAP3Kepsilon1 fusion protein using confocal microscopy and biochemical fractionation indicated that a substantial portion of the MAP3Kepsilon1 present in Arabidopsis cells is localized to the plasma membrane. Taken together, our results suggest that MAP3Kepsilon1 is required for the normal functioning of the plasma membrane in developing Arabidopsis pollen.

Isolation, characterization and culture of Thy1-positive cells from fetal rat livers.

AIM: To investigate whether Thy1 recognizes oval cells in the fetal liver and to characterize the cultured Thy1- selected cells from E14 rat livers. METHODS: Thy1 populations were analyzed by fluorescence activated cell sorter analysis. Thy1 positive cells were isolated using magnetic beads. Hepatic markers were detected by Western blotting, immunocytochemistry and RT-PCR. RESULTS: The percentage of Thy1-positive cells decreased during early development of fetal rat liver (E13-E16). E14 fetal livers contained 7.8% Thy1 positive cells, of which 61% were positive for alpha-fetoprotein (AFP) and 25% expressed albumin. The Thy1+ population expressed oval cell markers c-Kit and CXCR4, liver enriched-transcription factors HNF1alpha and HNF6, hepatocytic markers albumin, AFP and cytokeratin 18, and biliary marker cytokeratin 19. Thy1- selected cells formed only mesenchymal colonies when plated on collagen and in serum-containing media. Thy1 selected cells were able to form hepatic colonies positive for HNF1alpha, HNF6, albumin, AFP, cytokeratin 18, cytokeratin 19 and glycogen, when grown on STO feeder layers in serum free-media. CONCLUSION: Oval cells positive for Thy1 are present in early liver embryonic stages.

Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells.

We studied the proton secretion mechanisms involved with pHi regulation in immortalized rat proximal tubule cells (IRPTC), a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation on them. Using pHi measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC express both Na+/H+ exchanger and H+-ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24 h of serum starvation increased pHi from 7.08+/-0.008 (n=34) to 7.18+/-0.018 (n=33) as well as the pH recovery rate from intracellular acidification with NH4Cl from 0.29+/-0.022 pH U/min (n=14) to 0.50+/-0.024 pH U/min (n=14), without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, as the utilization of actinomycin D and cycloheximide significantly inhibited the upregulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO-126, significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity, whereas the putative p38 MAPK inhibitor SB-203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This upregulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.

The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20.

Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification and characterization.

Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism.

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.