HEMATOLOGIC DISEASES RESULTING FROM DEFECTS OF TYROSINE PHOSPHATASES

许多重要的过程都受到蛋白质酪氨酸磷酸化的调节， 受蛋白酪氨酸激酶(PTKs)和蛋白酪氨酸酶的调节 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，它们通过受体PTKs传递信号 或受体相关的PTKs。例如，菌落刺激因子1 (CSF1)，控制细胞增殖和分化的主要因素 巨噬细胞在细胞培养和体内，通过 CSFIR，一种受体PTK。异常的PTKs可导致造血功能衰竭或， 相反，过度增殖的疾病，如骨髓增生性疾病 综合症或白血病。慢性粒细胞白血病(CML)是由一种 使BCR和C-ABL融合的易位，产生融合蛋白， Bcr-abl，PTK活性异常。尽管我们已经学到了很多东西 关于PTKs如何调节正常的造血，以及异常的PTKs如何导致 疾病，对特定的PTP在正常或 病理生理学。这项研究的目的是定义 CSF1R信令和BCR-ABL中的两个PTP，SHPTP1和SHPTP2 转型。遗传和生化数据表明SHPTP1是阴性的 调节许多PTK通路，而SHPTP2可能发挥积极作用。 我们已经发现，来自蛾(甜瓜)小鼠的巨噬细胞缺乏 SHPTP1，在CSF1刺激下过度增殖。SHPTP1，过度增殖 以回应CSF1。SHPTP1似乎负向调节CSF1R信号 通过一种涉及底物招募到SHPTP1的新机制 催化结构域通过Grb2。我们还发现SHPTP1和SHPTP2结合 Bcr-abl，尽管它们可能具有不同的生化和生物学特征 后果。SHPTP2还与BCR-ABL中的97kD蛋白(P97)结合 转化的细胞可能与结合SHPTP2的蛋白质相同 在细胞因子刺激下。我们将定义SHPTP1的分子细节 CSFIR的监管。SHPTP1酪氨酸磷酸化位点(S) 对CSF1的反应将被映射和突变以防止酪氨酸 磷酸化和/或Grb2结合。这些突变体的特性将 在Me/Me中使用一种新的瞬时重建试验进行评估 巨噬细胞。结合SH2结构域的130kD Py蛋白 将对SHPTP1进行鉴定并确定其功能。是否全部 下游CSFIR通路同样受到SHPTP1丢失的影响 确定，以及我/我的倍增时间缩短的原因(S) 巨噬细胞。CSFIR途径对整个我/我的重要性 表型将通过转基因重组策略和 基因分析。这两个SHPTP在BCR-ABL上的结合位点为 并将阐明SHPTP2结合p97所需的结构域(S)。 不能结合SHPTP1或SHPTP1的BCR0ABL突变体的生物学特性 SHPTP2突变体无法结合P97，将在标准中进行测试 SHPTP-1的转化分析及恢复表达的影响 缺乏SHPTP1表达的K562细胞将被确定。最后，我们 将询问SHPTP/BCR-ABL关联的变化是否与CML相关 通过分析这些相互作用在母细胞和 慢性和急性期患者的中性粒细胞。这些研究应该 对PTP如何有助于控制正常 人类疾病中的造血及其破坏。

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.