SIGNAL TRANSDUCTION BY NON-TRANSMEMBRANE PTP S

非跨膜 PTP S 的信号转导

• 批准号: 2093188
• 负责人: BENJAMIN G. NEEL
• 金额: $ 30.77万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2093188
• 关键词: Drosophilidae; Schizosaccharomyces pombe; Xenopus; antisense nucleic acid; bioassay; biological signal transduction; growth factor receptors; keratinocyte; laboratory mouse; laboratory rabbit; membrane proteins; microinjections; molecular cloning; monoclonal antibody; mutant; neoplastic transformation; neutralizing antibody; phosphoprotein phosphatase; phosphorylation; protein isoforms; receptor binding; regulatory gene; skin neoplasms

Control of protein tyrosyl phosphorylation is an important mechanism for the regulation of cellular proliferation, the cell cycle, differentiation, and cell death. Disregulation of these control mechanisms can lead to neoplasia. Although much has been learned about how protein tyrosine kinases (PTK) help determine the steady state level of cellular phosphotyrosine, little is known about how phosphotyrosine phosphatases (PTPs) contribute to this process, and virtually nothing is known about how PTPs might contribute to disease. We will elucidate the biological roles of two nontransmembrane PTPs, PTP1B and SHPTP2, cloned during the first four years of this grant. Initial studies have implicated PTP1B in control of the cell cycle and in cell death pathways. In cycling cells, PTP1B resides in a high molecular weight complex on the cytosolic surface of the endoplasmic reticulum, and undergoes cell cycle- dependent modifications, including alternative splicing during G0 and G1, and new serine phosphorylation at G2/M. However, in human keratinocytes induced to undergo a terminal differentiation/cell death pathway by calcium ionophores, PTP1B is cleaved and released as an active fragment into the cytosol. This is reminiscent of a bacterial virulence determinant that kills cells by dephosphorylating tyrosol phosphorylated suggesting that PTP1B cleavage is important in keratinocyte cell death pathways. SHPTP2, the apparent human homologue of Drosophila csw, interacts directly with several ligand-activated growth factor receptor and becomes tyrosyl phosphorylated in response to growth factor addition, suggesting that it plays a critical role in early growth factor response. In this grant, neutralizing monoclonal antibodies against PTP1B will be generated to allow PTP1B mitotic phosphorylation sites will be mapped, and the biologic role of PTP1B in mitosis explored by microinjection of neutralizing antibodies and transient overexpression of wild type and phosphorylation site mutant in mammalian cells and S. pombe. The precise cleavage site of PTP1B in ionophore-treated keratinocytes will be defined and the role of PTP1B cleavage in inducing cell death assessed in normal cells and in skin cancers, which are refractory to induction of cell death pathways. The precise sites of interaction of SHPTP2 with growth factor receptors will be determined, and the effects of receptor binding and tyrosyl phosphorylation on SHPTP2 enzymatic activity assessed. PDGF receptor mutants that fail to bind SHPTP2 will be used to identify SHPTP2 downstream signaling targets. In collaborative studies, the ability to SHPTP2 to complement csw mutations will be determined, and if complementation is obtained, suppressor and potentiator screens in Drosophila will be used to suggest other components of SHPTP2 signal transduction pathways, Finally, a rapid in vitro bioassay for mapping SHPTP2 functional domains and targets will be developed using FGF-induced mesoderm induction in Xenopus as a model system. Since PTP1B and SHPTP2 appear to participate in control of all of the processes--the response to growth factors, cell cycle control, control of differentiation, and control of cell death--that are eluded by cancer cells, the results of the proposed studies should yield critical information into how PTPs help control these processes. Moreover, since tyrosyl phosphorylation is so commonly disregulated in cancer cells, this work should have important implication for understanding mechanisms of oncogenesis, and may suggest new therapeutic approaches to combat neoplasia.

控制蛋白酪酶磷酸化是重要机制 细胞增殖的调节，细胞周期， 分化和细胞死亡。 忽略这些控制 机制可以导致肿瘤。 虽然已经了解了很多 蛋白质酪氨酸激酶（PTK）如何有助于确定稳态水平 细胞磷酸酪氨酸的磷酸酪氨酸如何知之甚少 磷酸酶（PTP）有助于此过程，几乎没有什么是 了解PTP可能如何促进疾病。 我们将阐明 克隆的两个非跨膜PTP和SHPTP2的生物学作用 在这笔赠款的前四年。最初的研究有 在控制细胞周期和细胞死亡途径中，涉及PTP1B。 在循环细胞中，PTP1B驻留在高分子量复合物上 内质网的胞质表面，并经历细胞周期 - 依赖性修改，包括G0和G1期间的替代剪接 G2/m处的新丝氨酸磷酸化。 但是，在人角质形成细胞中 诱导通过 钙离子载体，PTP1B被切割并释放为活性片段 进入细胞质。 这让人想起细菌毒力 通过去磷酸化的酪醇磷酸化来杀死细胞的决定因素 表明PTP1B裂解在角质形成细胞死亡中很重要 途径。 SHPTP2，果蝇CSW的明显人类同源物， 与几种配体激活的生长因子受体直接相互作用 并因响应生长因子的添加而变成酪酶磷酸化， 表明它在早期生长因子反应中起着至关重要的作用。 在这笔赠款中，针对PTP1B的中和单克隆抗体将是 生成以允许PTP1B有丝分裂磷酸化位点的映射， PTP1B在微注射中探索的有丝分裂中的生物学作用 中和抗体和野生型的短暂过表达和 哺乳动物细胞和猪链球菌中的磷酸化位点突变体。 确切的 将定义经离子载体处理的角质形成细胞中PTP1B的切割位点 PTP1B裂解在正常情况下评估的细胞死亡中的作用 细胞和皮肤癌对诱导细胞的难治性 死亡道路。 SHPTP2与增长的相互作用的确切位置 将确定因子受体，以及受体结合的影响 评估了SHPTP2酶活性的酪酶磷酸化。 PDGF 无法结合SHPTP2的受体突变体将用于识别SHPTP2 下游信号目标。 在协作研究中 将确定SHPTP2以补充CSW突变，如果 获得互补，抑制器和增强器筛选 果蝇将用于建议SHPTP2信号的其他组件 转导途径，最后是用于映射的快速体外生物测定 SHPTP2功能域和目标将使用FGF诱导的 爪蟾中的中胚层诱导作为模型系统。 由于PTP1B和SHPTP2 似乎参与了所有过程的控制 - 回应 对于生长因素，细胞周期控制，分化的控制和 控制细胞死亡 - 被癌细胞避免了 拟议的研究应产生关键信息，以了解PTP的帮助 控制这些过程。 此外，由于酪酶磷酸化是如此 这项工作通常在癌细胞中不受控制，应该具有重要的 对理解肿瘤发生机制的影响，并可能表明 打击肿瘤的新治疗方法。