ROLE OF BAG2 AND SHP2 IN HEMATOPOIETIC SIGNALLING

许多重要的生物学过程受酪氨酰磷酸化的调节，酪氨酰磷酸化由蛋白酪氨酸激酶（PTK）和蛋白酪氨酸磷酸酶（PTPs）的相反作用控制。这些通路的异常调节可导致癌症等疾病。要完全理解酪氨酰磷酸化对细胞和整个生物体的调节，需要确定特定的PTKs和PTPs如何相互作用。这样的理解可能会导致开发新的，更具体的治疗人类疾病的治疗试剂。本研究计划的长期目标是了解造血细胞中含有SH 2结构域的酪氨酸磷酸酶SHP 2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的先前研究确立了SHP 2作为细胞因子受体、造血生长因子受体和多链免疫识别受体下游信号传导途径的重要组分。这些研究表明，一个97 kD的磷酸化酪氨酰蛋白是SHP 2作用的主要靶点/调节剂，但该蛋白的身份及其功能仍不清楚。在最初的资助期间，我们鉴定、纯化和克隆了p97。p97是Gab 1和果蝇Dos的亲戚，果蝇Dos是SHP-2同源物的调节器;因此，我们将p97重命名为Gab 2。对野生型和突变型Gab 2的影响的分析表明，它是细胞因子信号转导的关键调节因子，也参与其他类型造血细胞受体下游的信号传导。我们的研究表明，SHP 2在细胞因子受体信号转导中的作用不止一个步骤：一个需要Gab 2结合，另一个是Gab-2独立的。不依赖Ciab 2的途径是MAP激酶活化所需的，而Gab 2通过SHP 2起作用，调节MAP激酶活化下游/或平行于MAP激酶活化但在精氨酸诱导的基因表达上游的新信号传导途径。我们最近的研究表明，Gab 2通过一种新的涉及Shc的复合物被招募到细胞因子受体，然后为那些不直接结合PI 3 K的受体提供PI-3激酶激活的主要途径。我们将阐明细胞因子信号传导中Shc/Gab 2途径的细节。并确定Gab 2/PI 3 K复合物调节的其他途径。接下来，我们将定义SHP 2如何以Gab-2非依赖性方式调节MAP激酶激活，以及Gab 2/SHP 2复合物如何调节即刻早期基因转录。我们将表征“真无效”SHP 2敲除小鼠和Gab 2-/-小鼠的造血作用，以确定这些分子在体内的生理功能。这些研究应该对酪氨酰磷酸化是如何控制的产生新的见解。阐明特定的PTP如何有助于调节，并确定一个新的信号通路调节精氨酸诱导的基因表达。由于细胞因子信号传导的异常调节与白血病发生和某些癌症有关，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.