Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

The carboxyl-terminal (CT) domain of connexin43 (Cx43) has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST)-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.

Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Cyclic 3'-5' guanosine monophosphate-dependent activity in Leishmania amazonensis

Although there are some data concerning the nitric oxide and the cyclic 3'-5'guanosine monophosphate (cGMP) signaling pathway in trypanosomatids, there is no report about the cGMP-dependent enzymatic activity identification. In this sense, a cGMP dependent activity was detected on soluble fraction from Leishmania amazonensis promastigotes with a high metacyclic level. This information is valuable in order to explore the metabolic pathway of G kinase protein in this parasite.