Prekeris R, Hernandez R M, Mayhew M W, White M K, Terrian D M
Department of Anatomy and Cell Biology, East Carolina University School of Medicine, Greenville, North Carolina 27858, USA.
J Biol Chem. 1998 Oct 9;273(41):26790-8. doi: 10.1074/jbc.273.41.26790.
Protein kinase C-epsilon (PKC-epsilon) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-epsilon and actin. Full-length recombinant PKC-epsilon, but not PKC-betaII, -delta, -eta, or -zeta, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-epsilon amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-epsilon or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-epsilon colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-epsilon phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-epsilon sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-epsilon in a catalytically active conformation.
蛋白激酶C-ε（PKC-ε）含有一个假定的肌动蛋白结合基序，该基序是PKC基因家族中这个独特成员所特有的。我们利用缺失诱变来确定这个六肽基序对于PKC-ε与肌动蛋白的物理结合是否必要。全长重组PKC-ε，而非PKC-βII、-δ、-η或-ζ，以佛波酯依赖的方式与丝状肌动蛋白结合。缺失PKC-ε的222 - 230位氨基酸（包含一个假定的肌动蛋白结合基序）完全消除了这种结合活性。当用佛波酯处理过表达PKC-ε或该同工酶缺失突变体的NIH 3T3细胞时，只有野生型PKC-ε在细胞黏附区域与肌动蛋白共定位。在二元反应中，有可能证明纯化的丝状肌动蛋白能够直接刺激PKC-ε磷酸转移酶活性。这些以及其他发现支持这样的假说：PKC-ε序列中一个构象隐藏的肌动蛋白结合基序在该同工酶激活时会暴露，并在NIH 3T3成纤维细胞中作为一个主要的定位信号发挥作用。这种蛋白质-蛋白质相互作用足以使PKC-ε维持在催化活性构象。
