ROLE OF BAG2 AND SHP2 IN HEMATOPOIETIC SIGNALLING

许多重要的生物过程是由酪氨酸磷酸化调控的，而酪氨酸磷酸化是由蛋白酪氨酸激酶（PTKs）和蛋白酪氨酸磷酸酶（PTPs）的相反作用控制的。这些通路的异常调节可导致癌症等疾病。要完全理解细胞和整个生物体通过酪氨酸磷酸化的调节，需要确定特定的PTKs和PTPs如何相互作用。这种认识可能导致开发新的、更特异的治疗人类疾病的试剂。本研究计划的长期目标是了解造血细胞中含有SH2结构域的酪氨酸磷酸酶SHP2的生物学功能和作用机制。包括我们自己的实验室在内的几个实验室先前的研究表明，SHP2是细胞因子受体、造血生长因子受体和多链免疫识别受体下游信号通路的重要组成部分。这些研究表明，97kD磷酸酪氨酸蛋白是SHP2作用的主要靶点/调节因子，但该蛋白的身份及其功能尚不清楚。在最初的资助期内，我们鉴定、纯化和克隆了p97。p97是Gab1和果蝇Dos的近亲，是果蝇SHP-2同源基因的调节因子；因此，我们将p97重新命名为Gab2。对野生型和突变型Gab2影响的分析表明，它是细胞因子信号转导的关键调节剂，也参与其他类型造血细胞受体的下游信号传导。我们的研究证实，SHP2在细胞因子受体信号转导中不止一步起作用：一个需要Gab2结合，另一个是Gab2独立的。独立于ciab2的通路是MAP激酶激活所必需的，而Gab2通过SHP2起作用，调节MAP激酶激活下游或平行于MAP激酶激活，但在细胞因子诱导的基因表达上游的新信号通路。我们最近的研究表明，Gab2通过一个涉及Shc的新复合物被募集到细胞因子受体上，然后为那些不直接结合PI3K的受体提供了激活PI-3激酶的主要途径。我们将阐明细胞因子信号传导中Shc/Gab2通路的细节。并确定Gab2/PI3K复合物调节的其他途径。接下来，我们将定义SHP2如何以不依赖Gab2的方式调节MAP激酶激活，以及Gab2/SHP2复合物如何调节即时早期基因转录。我们将对“真零”SHP2敲除小鼠和Gab2-/-小鼠的造血作用进行表征，以确定这些分子在体内的生理功能。这些研究应该对如何控制酪氨酸磷酸化产生新的见解。阐明特异性ptp如何参与调控，并确定调节细胞因子诱导的基因表达的新信号通路。由于细胞因子信号的异常调节与白血病和一些癌症的发生有关，我们的研究也可能为人类肿瘤疾病的治疗干预提供新的靶点。

Many important biological processes are regulated by tyrosyl phosphorylation, which is controlled by the opposing actions of protein-- tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Abnormal regulation of these pathways can lead to diseases such as cancer. A complete understanding of cellular and whole organism regulation by tyrosyl phosphorylation requires defining how specific PTKs and PTPs interact. Such understanding may lead to the development of new, more specific therapeutic reagents for treating human disease. The long range goal of this research program is to understand the biological functions and mechanism of action of the SH2 domain-containing tyrosine phosphatase SHP2 in hematopoietic cells. Previous studies by several laboratories, including our own, established SHP2 as a vital component of signaling pathways downstream of cytokine receptors, hematopoietic growth factor receptors and multi- chain immune recognition receptors. These studies suggested that a 97kD phosphotyrosyl protein was a major target/regulator of SHP2 action, but the identity of this protein and its functions had remained unclear. During the initial funding period, we identified, purified and cloned p97. p97 is a relative of Gab1 and Drosophila Dos, a regulator of the fly homolog of SHP-2; thus, we renamed p97 as Gab2. Analyses of the effects of wild type and mutant versions of Gab2 suggest that it is a key regulator of cytokine signal transduction and also participates in signaling downstream of other types of hematopoietic cell receptors. Our studies establish that SHP2 acts at more than one step in cytokine receptor signal transduction: one requires Gab2 binding and the other is Gab-2 independent. The Ciab2-independent pathway is required for MAP kinase activation, whereas Gab2, acting via SHP2, regulates a novel signaling pathway downstream of/or parallel to MAP kinase activation but upstream of cytokine-induced gene expression. Our recent studies show that Gab2 is recruited to cytokine receptors via a novel complex involving Shc, and then provides the major route to PI-3 kinase activation for those receptors that do not bind PI3K directly. We will elucidate the details of the Shc/Gab2 pathway in cytokine signaling. and determine what other pathways are regulated by Gab2/PI3K complexes. Next, we will define how SHP2 regulates MAP kinase activation in a Gab-2- independent manner and how the Gab2/SHP2 complex regulates immediate early gene transcription. We will characterize the hematopoietic effects of "true-null" SHP2 knockout mice and Gab2-/- mice to determine the physiological function of these molecules in vivo. These studies should yield new insights into how tyrosyl phosphorylation is controlled. clarify how specific PTPs contribute to regulation, and identify a new signaling pathway regulating cytokine-induced gene expression. Since abnormal regulation of cytokine signaling is implicated in leukemogenesis and some carcinomas, our studies may also suggest new targets for therapeutic intervention in human neoplastic disease.