RELATED ADHESION KINASE IN MEGAKARYOCYTES

描述：(改编自申请者摘要)信号研究 血小板中的转导通路可能会提高我们对血小板的理解 在巨核细胞中的功能和这种研究可以提供关于 调节它们的生长和成熟。Avraham博士已经发现了一种 已知的人巨核细胞胞浆酪氨酸激酶 粘着斑酪氨酸激酶(RAFTK)。后者属于焦点 黏附蛋白激酶(FAK)基因家族是一种信号转导分子 对正常和肿瘤生长很重要。RAFTK还代表了一种常见的 整合素和有丝分裂原多肽作用的链接。人和小鼠 RAFTK的cDNAs已被克隆和鉴定。Xxxxce氨基酸 序列也表明RAFTK与FAK相似之处在于 巨核细胞相关基因将进一步表征 特别提到在信号转导中的作用。RAFTK将是 纯化、鉴定并研究其在信号转导中的作用。这个 RAFTK的功能域将被映射和结构-功能 关系已确定。将辨别RAFTK表达是否 是造血细胞增殖和分化所必需的。 在具体目标一中，将对RAFTK进行生化和 从免疫化学角度来看。RAFTK的CDNA编码区将在 大肠埃希菌。表达的重组蛋白将得到纯化。CDNA 编码完整RAFTK的基因将在杆状病毒系统中表达。这个 重组蛋白作为一种激酶的性质将被研究，并 将会产生针对重组RAFTK的单抗。抗体 识别RAFTK蛋白中不同的结构域将会得到发展。 将描绘RAFTK不同监管结构域的试剂将是 用于RAFTK蛋白及其蛋白的进一步生化鉴定 上游和下游衬底。在具体目标二中，参与 RAFTK蛋白在血小板活化及信号转导中的作用 巨核细胞的增殖将被描绘出来。这将涉及到 巨核细胞-骨髓成纤维细胞黏附的实验研究 巨核细胞和内皮细胞之间的相互作用。蛋白质 哪些与RAFTK相互作用将在这些实验中确定。在……里面 具体目标三将绘制RAFTK的功能域并 确定了蛋白质的结构-功能关系。数列 将确定RAFTK功能和本地化所需的组件。 对主要酪氨酸磷酸化位点(S)进行分析。富含脯氨酸的氨基 RAFTK中的酸序列将根据官能团定义 重要性。在具体目标四中，将确定RAFTK是否 表达是造血细胞增殖和分化所必需的。 RAFTK在胚胎干细胞中的基因位点将被破坏 同源重组。不能表达RAFTK的小鼠将 将被生成。造血缺陷小鼠的表型研究 将对发展进行审查。因此，在胚胎中进行靶向实验 干细胞将被设计来测试RAFTK在发育中的功能，作为 以及RAFTK在造血过程中的需求。

DESCRIPTION: (Adapted from Applicant's Abstract) Studies of signal transduction pathways in platelets may improve our comprehension of platelet function and such studies in megakaryocytes can provide information on regulation of their growth and maturation. Dr. Avraham has identified a cytoplasmic tyrosine kinase in human megakaryocytes known as Related Adhesion Focal Tyrosine Kinase (RAFTK). The latter belongs to the focal adhesion kinase (FAK) gene family and is a signal transduction molecule important for normal and neoplastic growth. RAFTK also represents a common link for the action of integrins and mitogenic peptides. Human and murine RAFTK CDNAS have been cloned and characterized. xxxxced amino acid sequences also indicate that RAFTK resembles FAK in that a The megakaryocyte-associated gene will be further characterized with particular reference to a role in signal transduction. The RAFTK will be purified and characterized and its role in signal transduction studied. The functional domains of RAFTK will be mapped and structure-function relationships determined. It will be discerned whether RAFTK expression is required for hematopoietic proliferation and differentiation. In Specific Aim I, RAFTK will be characterized biochemically and immunochemically. The CDNA, encoding domains of RAFTK will be expressed in E. coli. The recombinant proteins expressed will be purified. The CDNA which encodes the full RAFTK will be expressed in a Baculovirus system. The properties of the recombinant protein as a kinase will be studied and monoclonal antibodies to the recombinant RAFTK will be raised. Antibodies recognizing distinct domains in the RAFTK protein will be developed. Reagents which will delineate distinct regulatory domains of RAFTK will be used for further biochemical characterization of the RAFTK protein and its upstream and downstream substrates. In Specific Aim II, participation of RAFTK protein in signal transduction during platelet activation and megakaryocyte proliferation will be delineated. This will involve experiments measuring megakaryocyte-marrow fibroblast adhesion and studies of interactions between megakaryocytes and endothelial cells. Proteins which interact with RAFTK will be identified in these experiments. In Specific Aim III the functional domains of RAFTk will be mapped and structure-function relationships of the protein determined. Sequence components necessary for RAFTK function and localization will be identified. Major tyrosine phosphorylation site(s) will be analyzed. Proline-rich amino acid sequences in RAFTK will be defined with regard to functional importance. In Specific Aim IV, it will be determined whether RAFTK expression is required for hematopoietic proliferation and differentiation. The gene locus of RAFTK will be disrupted in embryonic stem cells by homologous recombination. Mice which are incapable of expressing RAFTK will be generated. The phenotype of mice with defects in hematopoietic development will be examined. Thus the targeting experiments in embryonic stem cells will be designed to test the function of RAFTK in development as well as the requirement for RAFTK in hematopoiesis.