NOVEL CYTOSKELETAL PKC BINDING PROTEIN AND SUBSTRATE

蛋白激酶Cs(PKCs)是一种可溶性的酶，也被发现 与细胞骨架有关。一种潜在的定向机制 PKC到特定的细胞骨架结构，如焦点接触和细胞- 细胞连接是通过与其他蛋白质的相互作用实现的。我们使用了一个 检测磷脂酰丝氨酸依赖的PKC相互作用的重叠试验 以确定可能介导PKC结合的蛋白质 细胞骨架结构。在许多情况下，通过这种方式确定的蛋白质 检测结果也表明是PKC底物。重叠试验为 用于筛选lambdagt11大鼠肾脏文库，以分离额外的和 新型PKC结合蛋白/底物。两个克隆，35A和35H，现在 被刻画出来了。两者都是PKC底物和结合蛋白，并且 磷酸化减少了PKC的结合。抗35H抗体识别An 培养肾上皮细胞中的80 kDa细胞骨架蛋白。35H 在细胞-细胞连接处与α-PKC共定位。佛波醇酯 刺激35H的快速磷酸化及其从 细胞-细胞连接以及广泛的膜褶皱。目的 这一建议的目的是利用PKC-35H相互作用的研究和 以~(35)H磷酸化为模型系统更好地确定 细胞骨架相关蛋白激酶的功能。

Protein kinase Cs (PKCs) are soluble enzymes that are also found associated with the cytoskeleton. One potential mechanism for targeting PKC to specific cytoskeletal structures such as focal contacts and cell- cell junctions is via interactions with other proteins. We have used an overlay assay that detects phosphatidylserine-dependent PKC interactions with other proteins to identify proteins that may mediate PKC binding to cytoskeletal structures. In many cases, the proteins identified by this assay have also been shown to be PKC substrates. The overlay assay was used to screen a lambdagt11 rat kidney library to isolate additional and novel PKC binding proteins/substrates. Two clones, 35A and 35H, have now been characterized. Both are PKC substrates and binding proteins, and phosphorylation decreases PKC binding. Antibodies to 35H recognize an 80 kDa cytoskeletal protein in cultured renal epithelial cells. 35H colocalizes with alpha-PKC at cell-cell junctions. Phorbol esters stimulate a rapid phosphorylation of 35H and its displacement from the cell-cell junctions as well as extensive membrane ruffling. The purpose of this proposal is to use the study of PKC-35H interactions and the effects of 35H phosphorylation as model systems to better define the functions of cytoskeletal-associated PKCs.