The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p.

The Saccharomyces cerevisiae Sln1 protein is a ''two-component'' regulator involved in osmotolerance. Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.     

Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta- glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.     

Dic2 and Dic3 loci confer osmotic adaptation and fungicidal sensitivity independent of the HOG pathway in Cochliobolus heterostrophus

Previously, we identified three gene loci, Dic1, Dic2, and Dic3, that confer high-osmolarity adaptation and dicarboximide/phenylpyrrole fungicide sensitivity in Cochliobolus heterostrophus. Dic1 encoded a group III histidine kinase, but the other genes were not characterized. In the present study, we revealed that both Dic2 and Dic3 are involved in the Skn7 pathway. Dic2 encoded an Skn7-type response regulator, ChSkn7. Strain N4502 contained D359N in the response regulator domain of ChSkn7. Strain E4503 contained a deletion of 50 amino acids in the DNA-binding domain. Strain N4507 was a null mutant of the ChSkn7 gene. All of the dic2 mutant strains showed similar levels of sensitivity to high osmolarity and similar levels of resistance to fungicides. These results strongly suggested that both the DNA-binding domain and response regulator domain are essential for Skn7 function in osmotic adaptation and fungicide sensitivity. A western blot analysis revealed that Dic3 is not involved in the regulation of Hog1-type MAPKs. The Chssk1/dic3 double mutant strains clearly showed greater resistance to fungicides than the single mutant strains. An additive effect was also observed in the high-osmolarity experiments. On the other hand, the dic3/dic2 double mutant strains did not show higher levels of resistance to fungicides and greater sensitivity to KCl than the single mutant strains. These results strongly suggested that the dic3 locus confer high-osmolarity adaptation and fungicide sensitivity independently from Ssk1-Hog1 pathway, but not the Skn7 pathway. Moreover, the dic3 strain and all dic2 strains showed similar levels of sensitivity to high-osmolarity stress and similar levels of resistance to fungicides, suggesting Dic3 to have an essential role in the Skn7 pathway. Our results provide new insight into the functions of the Skn7 pathway in filamentous fungi.     

Two-component response regulators Ssk1p and Skn7p additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus

Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungus unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the southern corn leaf blight fungus Cochliobolus heterostrophus. Both ChSsk1- and ChSkn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The Chssk1 Chskn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1- and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.     

BRD7调控Rb/E2F通路的分子机制研究

BRD7是采用cDNA代表性差异分析法克隆的一个新的Bromodomain基因,过表达BRD7可抑制鼻咽癌细胞的生长和细胞周期进程,同时发现BRD7基因可以调控Rb/E2F通路的活性.该研究旨在进一步探讨BRD7调控Rb/E2F通路的分子机制.通过蛋白质印迹和RT-PCR实验方法发现,BRD7能够降低Rb的磷酸化水平,抑制cyclinD1、cyclinE的蛋白质表达,上调CDK4抑制子P19的mRNA表达,但对CDK4和CDK2的蛋白质表达没有明显影响;通过荧光素酶实验从转录调控水平进一步证实了BRD7能够明显抑制cyclinD1启动子活性;采用反义核酸技术抑制COS7细胞内源性BRD7的表达后,发现cyclinD1、cyclinE、磷酸化Rb的蛋白质表达水平上调,并且可以促进细胞生长.这些结果表明:BRD7参与调控Rb/E2F信号通路中重要靶分子的表达,抑制Rb/E2F通路的活性,从而阻止细胞周期G1-S期进程,抑制鼻咽癌细胞生长.     

Age-related decrease of protein kinase G activation in vascular smooth muscle cells

The p21 (cip1/waf1) protein induces cell cycle arrest through inhibition of the activity of cdk (cyclin dependent kinase)/cyclin complexes. Expression of p21 is induced in a p53-dependent manner by DNA damage. p21 can also be induced independently of p53 by phorbol ester or okadaic acid. In this study, we have addressed the role of the PKC (protein kinase C) signaling pathway in the induction of p21 in response to PMA (phorbol myristate acetate) and okadaic acid. Levels of p21 (protein and mRNA) rapidly increased (within approximately 4 h) in U937 cells treated with PMA. The PKC-specific inhibitors RO 31-8220 and GF109203X down-regulated PMA or okadaic acid-induced p21 expression. Following persistent PKC activation, p21 mRNA levels remained elevated, indicating an enhanced stability of the mRNA. Using actinomycin D to measure mRNA stability and p21 promoter luciferase assays to measure activity, we provide evidence to support a role for the PKC signaling pathway in p21 mRNA stability. Thus, PKC regulates the amount of p21 in U937 cells at the level of mRNA accumulation and translation.