Cloning and Characterization of Protein Tyrosine Kinases

该项目涉及从自然杀伤细胞克隆的蛋白酪氨酸激酶（PTK）的持续表征。该项目的主要重点是研究与羧基末端Src激酶（Csk）具有显著同源性的PTK; Csk同源激酶Chk（以前称为Lsk）。在发现Chk之前，Csk是已知的唯一磷酸化Src家族激酶的保守羧基端酪氨酸并下调其催化活性的PTK。与普遍表达的Csk不同，Chk主要在造血细胞中表达。我们已经表明，Chk的表达是可诱导的T细胞和外周血单核细胞。然而，我们的研究表明，与Csk不同，Chk表达不会抑制T细胞受体功能。为了了解Csk和Chk在T细胞中的不同作用，我们从T细胞裂解物中鉴定了Chk Src同源（SH）2结构域结合蛋白。今年，我们发表了一些研究，确定桩蛋白是一种Chk结合蛋白。此外，筛选具有Chk SH 3结构域的噬菌体展示文库产生富含脯氨酸的肽，其可以代表Chk SH 3配体。计算机搜索已经确定了可能的蛋白质候选人作为Chk相互作用蛋白。这些线索正在调查中。加上我们对Chk表达的持续研究和对Chk敲除小鼠的调查，这项工作应该有助于了解巨噬细胞和/或T细胞和NK细胞粘附和免疫调节的分子控制。

This project involves the continued characterization of protein tyrosine kinases (PTK) cloned from natural killer cells. The primary emphasis of this project is the study of a PTK that has significant homology to the carboxyl-terminal Src kinase (Csk); the Csk homolgous kinase, Chk (previously known as Lsk). Before the discovery of Chk, Csk was the only PTK known to phosphorylate the conserved carboxyl-terminal tyrosine of Src family kinases and down-regulate their catalytic activity. Unlike Csk, which is ubiquitously expressed, Chk is expressed primarily in hematopoietic cells. We have shown Chk expression to be inducible in both T cells and peripheral blood monocytes. However, our studies have shown that unlike Csk, Chk expression does not inhibit T cell receptor function. In an effort to understand the different roles of Csk and Chk in T cells, we have identified Chk Src homology (SH)2 domain binding proteins from T cell lysates. This year we published studies identifying paxillin as a Chk binding protein. In addition, screening of a phage display library with the Chk SH3 domain yielded a proline rich peptide that may represent a Chk SH3 ligand. Computer searches have identified possible protein candidates as Chk interaction protiens. These leads are now under investigation. Together with our continuing studies of the expression of Chk and investigation of Chk knockout mice, this work should shed light on the molecular control of macrophage and/or T cell and NK cell adherence and immune regulation.