HEMATOLOGIC DISEASES RESULTING FROM DEFECTS OF TYROSINE PHOSPHATASES

许多重要的过程都受到蛋白酪氨酸磷酸化的调节， 其由蛋白质-酪氨酰激酶（PTKs）和蛋白质-酪氨酰 磷酸酶（PTP）。 在造血过程中，体内平衡是由 生长因子和细胞因子的作用，其通过受体PTKs进行信号传导 或受体相关的PTK。 例如，集落刺激因子1 CSF 1是控制细胞增殖和分化的主要因子 巨噬细胞在细胞培养和体内，通过传递信号， CSFlR，PTK受体。 异常的PTK可导致造血功能衰竭， 相反，过度增殖疾病，如骨髓增生性疾病， 综合征或白血病。 慢性粒细胞白血病（CML）是由一种 融合BCR与C-ABL的易位，产生融合蛋白， BCR-ABL，PTK活性失调。 尽管我们知道 关于PTKs如何调节正常的造血，以及异常的PTKs如何导致 疾病，很少有人知道的作用，具体的PTP在正常或 病理生理学 本研究的目的是确定 CSF 1 R信号传导和BCR-ABL中的两种PTP SHPTP 1和SHPTP 2 转型 遗传和生化数据表明SHPTP 1是阴性的。 SHPTP 2是许多PTK通路的调节因子，而SHPTP 2可能起积极作用。 我们已经发现，来自Motheaten（Melme）小鼠的巨噬细胞， SHPTP 1，响应CSF 1过度增殖。 SHPTP 1，过度增殖 对CSF 1的响应。 SHPTP 1似乎负调节CSF 1 R信号传导 通过一种新的机制，包括招募底物到SHPTP 1 通过GRB 2催化结构域。 我们还发现SHPTP 1和SHPTP 2结合 BCR-ABL，尽管它们可能具有不同的生化和生物学特性， 后果 SHPTP 2还与BCR-ABL中的97 kD蛋白（P97）结合 转化的细胞，其可能与结合SHPTP 2的蛋白相同 在细胞因子刺激下。 我们将定义SHPTP 1的分子细节， CSFIR的监管。 SHPTP 1酪氨酸磷酸化的位点 对CSF 1的反应将被定位和突变，以防止酪氨酰 磷酸化和/或GRB 2结合。 这些突变体的特性将 在我/我中使用新型瞬时重建测定进行评估 巨噬细胞 结合人的SH 2结构域的130 kD pY蛋白， 将鉴定SHPTP 1并确定其功能。 是否所有 下游CSFIR通路同样受到SHPTP 1缺失的影响， 确定，以及我/我的倍增时间缩短的原因 巨噬细胞 CSFIR途径对整体ME/ME的重要性 表型将通过转基因重建策略和 遗传分析 BCR-ABL上两种SHPTP的结合位点将是 将阐明结合p97所需的SHPTP 2结构域。 BCR 0ABL突变体不能结合SHPTP 1或 不能结合P97的SHPTP 2突变体将在标准品中进行检测 转化试验以及恢复SHPTP-1表达对 将测定缺乏SHPTP 1表达的k562细胞。 最后我们 我将询问SHPTP/BCR-ABL关联的变化是否与CML相关 通过测定这些相互作用在原始细胞中的状态， 来自慢性期和急性期患者的中性粒细胞。 这些研究应 产生新的见解如何PTPs有助于控制正常的 造血及其在人类疾病中破坏。

Many important processes are regulated by protein-tyrosy phosphorylation, which is regulated by protein-tyrosyl kinases (PTKs) and protein-tyrosyl phosphatases (PTPs). In hematopoiesis, homeostasis is maintained by the actions of growth factors and cytokines, which signal through receptor PTKs or receptor-associated PTKs. For example, colony stimulating f actor 1 (CSF1), the major factor controlling the proliferation and differentiation of macrophages in cell culture and in vivo, transmits signals through the CSFIR, a receptor PTK. Abnormal PTKs can cause hematopoietic failure or, conversely, diseases of hyper-proliferation, such as myeloproliferative syndromes or leukemia. Chronic myelogenous leukemia (CML) results from a translocation that fuses BCR with C-ABL, resulting in a fusion protein, BCR-ABL, with dysregulated PTK activity. Although much as been learned about how PTKs regulate normal hematopoiesis, and how abnormal PTKs cause disease, little is known about the roles of specific PTPs in normal or patho-physiology. The goal of this research is to define the function of two PTPs, SHPTP1 and SHPTP2, in CSF1R signaling and in BCR-ABL transformation. Genetic and biochemical data suggest SHPTP1 is a negative regulator of many PTK pathways, whereas SHPTP2 may play a positive role. We have found that macrophages from motheaten (melme) mice, which lack SHPTP1, hyper-proliferate in response to CSF1. SHPTP1, hyper-proliferate in response to CSF1. SHPTP1 appears to negatively regulate CSF1R signaling by a novel mechanism involving recruitment of substrates to the SHPTP1 catalytic domain via GRB2. We have also found that SHPTP1 and SHPTP2 bind BCR-ABL, although they likely have distinct biochemical and biological consequences. SHPTP2 also binds to a 97kD protein (P97) in BCR-ABL transformed cells which may be identical to a protein that binds SHPTP2 upon cytokine stimulation. We will define the molecular details of SHPTP1 regulation of the CSFIR. The site(s) of SHPTP1 tyrosyl phosphorylation in response to CSF1 will be mapped and mutated to prevent tyrosyl phosphorylation and/or GRB2 binding. Thr properties of these mutants will be assessed using a novel transient reconstitution assay in me/me macrophages. The 130 kD pY-protein that binds to the SH2 domains of SHPTP1 will be identified and its function determined. Whether all downstream CSFIR pathways are equally affected by loss of SHPTP1 will be ascertained, as will the reason(s) for the shortened doubling time in me/me macrophages. The importance of the CSFIR pathway to the overall me/me phenotype will be assessed by a transgenic reconstitution strategy and by genetic analysis. The binding sites for both SHPTPs on BCR-ABL will be mapped and the domain(s) of SHPTP2 required to bind p97 will be elucidated. The biological properties of BCR0ABL mutants unable to bind SHPTP1 or SHPTP2 mutants unable to bind P97, will be tested in standard transformation assays and the effects of restoring SHPTP-1 expression to k562 cells, which lack SHPTP1 expression, will be determined. Finally, we will ask whether changes in SHPTP/BCR-ABL associations correlate with CML progression by assaying the status of these interactions in blasts and neutrophils from chronic and acute phase patients. These studies should yield new insights into how PTPs contribute to the control of normal hematopoiesis and its disruption in human disease.