Design and characterization of a traceable protein kinase Cα
Design and characterization of a traceable protein kinase Cα
复制标题
DOI：
10.1021/bi0622017
复制
发表时间：
2007-03-06
期刊：
影响因子：
2.9
通讯作者：
Rotenberg, Susan A.
中科院分区：
文献类型：
--
作者：
Abeyweera, Thushara P.;Rotenberg, Susan A.
蛋白激酶C α (PKC α)是控制癌症相关表型（如侵袭和增殖）途径的关键成分。作为PKC α直接底物的蛋白质为抗癌药物设计提供了潜在的靶点。为了鉴定特异性底物，在ATP结合位点构建了PKC α突变体（M417A），使其能够结合通过腺苷N-6氨基衍生的立体大ATP类似物（[γ -p -32]-N-6-苯基-ATP）。由于这种类似物可以被突变激酶利用，但不能被野生型PKC α（或可能是其他蛋白激酶）磷酸化肽或蛋白底物，p -32标记的产物是突变PKC α的直接产物。用[γ -p -32]- n -6-苯基ATP进行的动力学分析表明，突变体对肽底物的亲和力保持不变（K-m = 12.4 μ M）， V-max值（10.3 pmol/min）仅比使用天然ATP的野生型酶低3倍。然而，与[γ - p -32]ATP相比，突变体在体外的亲和力（K-m = 82.8 μ M）略低于野生型酶（K-m = 9.3 μ M），但能够在非运动性MCF-10A人乳腺细胞（内源性ATP）中引起侵袭性运动，如先前描述的野生型PKC α。flag标记的PKC α突变体在MCF-10A细胞中表达，并用于从裂解物中共免疫沉淀高亲和力底物。免疫微球与[γ -p -32]- n -6-苯基- atp反应，通过SDS-PAGE和放射自显影分析放射性标记产物。选定波段的质谱鉴定了PKC的几种已知底物，从而验证了这些研究中使用的方法。这些发现为这种可追溯的PKC α突变体的未来应用提供了基础。
Protein kinase C alpha (PKC alpha) is a critical component of pathways that govern cancer-related phenotypes such as invasion and proliferation. Proteins that serve as immediate substrates for PKC alpha offer potential targets for anticancer drug design. To identify specific substrates, a mutant of PKC alpha (M417A) was constructed at the ATP binding site such that it could bind a sterically large ATP analogue derivatized through the N-6 amino group of adenosine ([gamma-P-32]-N-6-phenyl-ATP). Because this analogue could be utilized by the mutant kinase but not by wild-type PKC alpha (or presumably other protein kinase) to phosphorylate peptide or protein substrates, P-32-labeled products were the direct result of the mutant PKC alpha. Kinetic analysis with [gamma-P-32]-N-6-phenyl-ATP revealed that the mutant retained undiminished affinity for the peptide substrate (K-m = 12.4 mu M) and a V-max value (10.3 pmol/min) that was only 3-fold lower than that exhibited by the wild-type enzyme with natural ATP. However, with [gamma-P-32]ATP, the mutant had a somewhat lower affinity (K-m = 82.8 mu M) than the wild-type enzyme (K-m = 9.3 mu M) in vitro but was competent in causing aggressive motility in nonmotile MCF-10A human breast cells (with endogenous ATP), as previously described for wild-type PKC alpha. The FLAG-tagged PKC alpha mutant was expressed in MCF-10A cells and used to co-immunoprecipitate high-affinity substrates from lysates. Immunopellets were reacted with [gamma-P-32]-N-6-phenyl-ATP, and radiolabeled products were analyzed by SDS-PAGE and autoradiography. Mass spectrometry of selected bands identified several known substrates of PKC, thereby validating the methods used in these studies. These findings provide a foundation for future applications of this traceable PKC alpha mutant.