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植物转脂蛋白(LTPs)是多基因编码的蛋白家族,广泛分布于高等植物.虽然LTPs的确切功能至今仍不完全清楚,但它参与植物生物、非生物胁迫反应以及它的抗性功能已成为近年来的研究热点.关于LTPs功能的调节机制目前几乎一无所知.最近,从白菜中分离的钙调素结合蛋白-10(CaMBP-10)被鉴定为植物转脂蛋白家族成员,并且,体外实验证明钙调素(CaM)调节其脂质结合活性.为了深入了解转脂蛋白功能的调节机制,本文研究了CaMBP-10的磷酸化作用,发现CaMBP-10可被豌豆质膜内源性蛋白激酶磷酸化,钙离子(Ca2+)能刺激磷酸化,钙螯合剂EGTA以及CaM拮抗剂W-7和TFP均能显著抑制磷酸化.免疫印迹分析最终确定该激酶为CDPK家族成员.构建突变体进一步研究了CaMBP-10的磷酸化位点,发现其位于蛋白的C-末端区域,并与已确定的CaM结合位点重合.同时,分析结果表明CaM能抑制CaMBP-10的磷酸化.反之,CaMBP-10的磷酸化又能阻断其与CaM的结合,显示出两种调节方式相互竞争的特点.为深入研究磷酸化作用对CaMBP-10脂质结合活性的影响,构建突变体(Ser83Asp,Ser85Asp)以模拟磷酸化状态.实验结果显示,磷酸化作用能显著增强CaMBP-10的脂质结合活性,而且突变体的脂质结合活性不受CaM的影响.采用胶内磷酸化测定法(in-gelkinaseassay)研究了激酶的自磷酸化特点以及CaMBP-10对激酶自磷酸化的影响,发现CaMBP-10能激活激酶的自磷酸化,激酶的自磷酸化又能促进其对底物的磷酸化作用.这样,激酶的自磷酸化与底物的磷酸化形成一种“正反馈环”的调节模式.综合研究结果,本文首次证明了LTP受CaM结合和CDPK磷酸化的双重调节.而且,CaM结合位点与磷酸化位点的重合预示可能存在特殊的调节机制,以协同应答胞内的Ca2+信号.
Plant lipoproteins (LTPs) are multi-gene-encoded protein families widely distributed in higher plants.Although the exact function of LTPs is still not fully understood, its involvement in plant biotic and abiotic stress responses and its resistance function have become Recently, there is little research on the regulation mechanism of LTPs function.Recently, calmodulin-binding protein-10 (CaMBP-10) isolated from Chinese cabbage was identified as a member of plant lipoprotein family, In vitro experiments show that calmodulin (CaM) regulates its lipid binding activity.In order to further understand the regulatory mechanism of the function of the lipoprotein, this paper studied the phosphorylation of CaMBP-10 and found that CaMBP-10 can be endogenous to pea plasma membrane Phosphorylation of protein kinase, calcium ion (Ca2 +) stimulated phosphorylation, calcium chelator EGTA and CaM antagonist W-7 and TFP both inhibited the phosphorylation significantly. Western blot analysis confirmed that the kinase is a CDPK family member. The phosphorylation site of CaMBP-10 was further studied and found to be located in the C-terminal region of the protein and coincident with the established CaM binding site.At the same time, CaMP inhibited the phosphorylation of CaMBP-10, CaMBP -10 phosphorylation can block its binding to CaM, showing the two regulatory modes compete with each other.In order to further study the phosphorylation of CaMBP-10 lipid binding activity, the construction of mutants (Ser83Asp, Ser85Asp ) To simulate the phosphorylation state.Experimental results show that phosphorylation can significantly enhance the lipid-binding activity of CaMBP-10, and the lipid-binding activity of the mutant is not affected by CaM.The in vivo phosphorylation assay (in- gelkinaseassay) to study the autophosphorylation of kinases and the effect of CaMBP-10 on the autophosphorylation of kinases. We found that CaMBP-10 activates autophosphorylation of kinases, which in turn promotes autophosphorylation of the substrates .In this way, the autophosphorylation of kinase and substrate phosphorylation form a “positive feedback loop ” regulation mode.A comprehensive study, we first demonstrated that LTP by CaM binding and CDPK phosphorylation double regulation.Moreover, The coincidence of the CaM binding site with the phosphorylation site indicates that there may be a special regulatory mechanism to synergistically respond to intracellular Ca2 + signaling.