NOVEL CYTOSKELETAL PKC BINDING PROTEIN AND SUBSTRATE

蛋白激酶C（PKC）是可溶性酶，也被发现 与细胞骨架有关。 一种潜在的瞄准机制 PKC与特定的细胞骨架结构，如局灶性接触和细胞- 细胞连接是通过与其他蛋白质的相互作用。 我们使用了一个 检测磷脂酰丝氨酸依赖性PKC相互作用的覆盖试验 与其他蛋白质，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，通过这种方法鉴定的蛋白质 测定也已显示是PKC底物。 覆盖试验为 用于筛选大鼠肾脏文库以分离额外的和 新的PKC结合蛋白/底物。 两个克隆体，35 A和35 H， 被定性。 两者都是PKC底物和结合蛋白， 磷酸化降低PKC结合。 抗35 H抗体可识别 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35 H的快速磷酸化及其从细胞膜上的置换， 细胞-细胞连接以及广泛的膜皱褶。 目的 这项建议的一个重要方面是利用PKC-35 H相互作用的研究和 35 H磷酸化的影响作为模型系统，以更好地定义 细胞膜相关PKC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.