Zähringer H, Holzer H, Nwaka S
Institut für Biochemie und Molekularbiologie, Universität Freiburg, Germany.
Eur J Biochem. 1998 Aug 1;255(3):544-51. doi: 10.1046/j.1432-1327.1998.2550544.x.
In Saccharomyces cerevisiae cAMP-dependent protein kinase (cAPK) is involved in nutrient sensing and growth regulation via the Ras/cAMP pathway. Target enzymes, e.g. neutral trehalase, are activated or inactivated rapidly by cAPK-mediated phosphorylation. In addition, stress-induced transcription of genes of the general stress-response, e.g. HSP12, is negatively regulated via cAPK. We have investigated the effect of low cAPK activity on the stress-induced expression of neutral trehalase Nth1p. For this purpose we used mutants (tpk1tpk2TPK3, tpk1TPK2tpk3 and TPK1tpk2tpk3) with double knockouts of the three TPK genes encoding catalytic subunits of cAPK. It is shown that the tpk1tpk2TPK3 mutant, which has very low cAPK activity, exhibits a heat-stress-induced inactivation of neutral trehalase that is not observed in tpk1TPK2tpk3, TPK1tpk2tpk3 mutants and wild-type cells. However, heat stress induces an increase in NTH1 mRNA in the tpk1tpk2TPK3 mutant. Introduction of a plasmid carrying the TPK1 or TPK2 gene into tpk1tpk2TPK3 cells restores the heat-induced increase of neutral trehalase activity. In vitro and in vivo results suggest that the heat induced inactivation of neutral trehalase is due to a reversible inactivation of Nth1p. Our data indicate that a certain level of phosphorylation is essential for maintenance of neutral trehalase activity during heat shock in S. cerevisiae. Two identical putative cAPK phosphorylation sites have been found in the sequence predicted for the Nth1p. Stabilization and activation of neutral trehalase may be regulated by these sites. Furthermore, our data suggest that the heat-stress-induced transcription of the NTH1 gene is not negatively regulated by cAPK, that the TPK genes have no effect on the glucose repression of the NTH1 gene, and that non-detectable neutral trehalase activity in derepressed tpk1tpk2TPK3 cells is correlated with the reduced thermotolerance observed in this strain, similar to the heat-shock-recovery defect reported for the nth1delta mutant.
在酿酒酵母中，环磷酸腺苷依赖性蛋白激酶（cAPK）通过Ras/环磷酸腺苷途径参与营养感知和生长调节。靶酶，如中性海藻糖酶，可通过cAPK介导的磷酸化迅速被激活或失活。此外，一般应激反应相关基因（如HSP12）的应激诱导转录通过cAPK受到负调控。我们研究了低cAPK活性对中性海藻糖酶Nth1p应激诱导表达的影响。为此，我们使用了三个编码cAPK催化亚基的TPK基因双敲除的突变体（tpk1tpk2TPK3、tpk1TPK2tpk3和TPK1tpk2tpk3）。结果表明，cAPK活性非常低的tpk1tpk2TPK3突变体在热应激诱导下会出现中性海藻糖酶失活，而在tpk1TPK2tpk3、TPK1tpk2tpk3突变体和野生型细胞中未观察到这种现象。然而，热应激会诱导tpk1tpk2TPK3突变体中NTH1 mRNA增加。将携带TPK1或TPK2基因的质粒导入tpk1tpk2TPK3细胞可恢复热诱导的中性海藻糖酶活性增加。体外和体内结果表明，热诱导的中性海藻糖酶失活是由于Nth1p的可逆失活。我们的数据表明，一定水平的磷酸化对于酿酒酵母在热休克期间维持中性海藻糖酶活性至关重要。在预测的Nth1p序列中发现了两个相同的假定cAPK磷酸化位点。中性海藻糖酶的稳定和激活可能受这些位点调控。此外，我们的数据表明，NTH1基因的热应激诱导转录不受cAPK负调控，TPK基因对NTH1基因的葡萄糖抑制没有影响，并且在去阻遏的tpk1tpk2TPK3细胞中检测不到的中性海藻糖酶活性与该菌株中观察到的耐热性降低相关，类似于nth1delta突变体报道的热休克恢复缺陷。
