Rapid in vitro conformational changes of the catalytic site of PKC alpha assessed by FIM-1 fluorescence.
Rapid in vitro conformational changes of the catalytic site of PKC alpha assessed by FIM-1 fluorescence.
复制标题
通过 FIM-1 荧光评估 PKC α 催化位点的体外快速构象变化。
作者：
A. Janoshazi;J. de Barry
为了研究蛋白激酶C(PKCalpha)的激活过程，我们使用了一种荧光探针FIM-1，它是一种双吲哚马来酰亚胺衍生物，它与催化结构域上的ATP结合位点结合[Chen，C.S.，and Poenie，M.(1993)J.Biol]。化学。268,15812]。这使得我们能够在体外直接观察激活过程中ATP结合位点的微环境。PKCalpha与FIM-1的结合亲和力(EC(50)在6~10 nM之间)既不受PKCalpha激活条件的影响，也不受酶降解作用的影响。PKCalpha-FIM-1复合物的荧光产率取决于PKCalpha的激活状态。这种荧光产量在蛋白降解时被降低，这使得我们能够研究Mu-calain对PKC蛋白的降解速率及其辅因子的修饰。在部分活化的PKCalpha上还观察到两个结合钙的部位。佛波酯(TPA)作用后，PKC的活化呈双指数动力学特征，包括5min内完成的快速阶段和持续至少30min的缓慢阶段，反映了几个激活步骤。TPA在膜结合的PKCalpha上有两个不同的结合部位(EC(50)=31+/-12和580+/-170 nM)，并研究了磷脂酰丝氨酸和钙离子对它们的调节作用。高亲和力的TPA结合部位高度保守，甚至在可溶性酶上也是如此。我们的研究表明，低浓度的TPA结合会触发可溶性PKCalpha的构象变化，从而影响其催化区的微环境。
To study the activation process of protein kinase C (PKCalpha), we used a fluorescent probe, FIM-1, a bis-indolylmaleimide derivative, which binds to the ATP-binding site on the catalytic domain [Chen, C. S., and Poenie, M. (1993) J. Biol. Chem. 268, 15812]. This enabled us to directly observe the microenvironment of the ATP-binding site in vitro during the activation process. The FIM-1 binding affinity for PKCalpha (EC(50) between 6 and 10 nM) was affected neither by PKCalpha activating conditions nor by enzyme proteolysis. The fluorescence yield of the PKCalpha-FIM-1 complex depended on the PKCalpha activation state. This fluorescence yield was decreased upon proteolysis, which allowed us to study the rate of PKC proteolysis by mu-calpain and its modification by cofactors. Two binding sites were also observed for Ca2+ on the partially activated PKCalpha. After phorbol ester (TPA) application, PKC activation was characterized by biexponential kinetics, including a rapid phase completed within 5 min and a slow phase lasting at least 30 min, which reflected several activation steps. Two different binding sites for TPA were revealed on membrane-associated PKCalpha (EC(50) = 31 +/- 12 and 580 +/- 170 nM), and their modulation by phosphatidylserine and Ca2+ was characterized. The high-affinity TPA binding site was highly conserved, even on the soluble enzyme. Our study shows that binding of low concentrations of TPA triggers conformational changes in the soluble PKCalpha, which affect the microenvironment of its catalytic domain.