Osuna Lidia, Coursol Sylvie, Pierre Jean-Noël, Vidal Jean
Laboratorio de Fisiologia Vegetal, Facultad de Biologia, Universidad de Sevilla, Avda., Reina Mercedes, Sevilla, Spain.
Biochem Biophys Res Commun. 2004 Feb 6;314(2):428-33. doi: 10.1016/j.bbrc.2003.12.103.
In mesophyll cells (MC) of Digitaria sanguinalis, the C(4)-phosphoenolpyruvate carboxylase (C(4)-PEPC) initiating the photosynthetic pathway is controlled by a complex light-dependent phosphorylation process. We showed previously that the transduction cascade involves the phosphoinositide pathway and a Ca(2+)-dependent step, which precedes the upregulation of the PEPC kinase (PEPCk). We have now further characterized the cascade component requiring Ca(2+). A Ca(2+)-dependent protein kinase that shows several characteristics of the conventional type of mammalian protein kinase C (PKC) was detected in protein extracts from mesophyll cell protoplasts (MCPs). It catalyzed the in vitro phosphorylation of the C1-peptide PKC substrate and was markedly inhibited by a PKC-specific pseudosubstrate domain. However, it was only modestly activated by the phospholipids phosphatidylserine and lysophosphatidylcholine, while choline, oleyl acetylglycerol, phosphatidylinositol, and the phorbol ester phorbol 12-myristate 13-acetate did not show any effect. Nevertheless, its activity was found to be associated with a polypeptide of 75kDa that was recognized by a PKC antibody raised against the C-terminus of rabbit PKCbeta II. In addition, this protein kinase was also inhibited by the Ca(2+)-dependent protein kinase (CDPK)/PKC inhibitors W7, H7, and staurosporine. Surprisingly, it was found to be phosphorylated in dark-adapted MCPs, albeit to a low extent, and this did not change during protoplast induction by light. W7, H7, and staurosporine were shown to markedly inhibit C(4)-PEPC phosphorylation in light-treated MCPs. These results support the view that this protein kinase is a good candidate to represent the Ca(2+)-activated component of the C(4)-PEPC phosphorylation cascade.
在马唐叶肉细胞（MC）中，启动光合途径的C4-磷酸烯醇式丙酮酸羧化酶（C4-PEPC）受一个复杂的光依赖磷酸化过程调控。我们之前表明，转导级联涉及磷酸肌醇途径和一个Ca2+依赖步骤，该步骤先于PEPC激酶（PEPCk）的上调。我们现在进一步表征了需要Ca2+的级联组分。在叶肉细胞原生质体（MCP）的蛋白质提取物中检测到一种Ca2+依赖蛋白激酶，它表现出传统类型的哺乳动物蛋白激酶C（PKC）的几个特征。它催化C1-肽PKC底物的体外磷酸化，并被PKC特异性假底物结构域显著抑制。然而，它仅被磷脂酰丝氨酸和溶血磷脂酰胆碱适度激活，而胆碱、油酰乙酰甘油、磷脂酰肌醇和佛波酯佛波醇12-肉豆蔻酸13-乙酸酯没有任何作用。尽管如此，其活性被发现与一个75kDa的多肽相关，该多肽被针对兔PKCβII C末端产生的PKC抗体识别。此外，这种蛋白激酶也被Ca2+依赖蛋白激酶（CDPK）/PKC抑制剂W7、H7和星形孢菌素抑制。令人惊讶的是，发现在暗适应的MCP中它会被磷酸化，尽管程度较低，并且在光诱导原生质体过程中这一情况没有改变。W7、H7和星形孢菌素被证明能显著抑制光处理的MCP中C4-PEPC的磷酸化。这些结果支持这样一种观点，即这种蛋白激酶是代表C4-PEPC磷酸化级联中Ca2+激活组分的一个良好候选者。
