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RELATED ADHESION KINASE IN MEGAKARYOCYTES

巨核细胞中的相关粘附激酶

基本信息

批准号：
6184124
负责人：
SHALOM AVRAHAM
金额：
$ 38.47万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-08-01 至 2001-06-30
项目状态：
已结题

项目摘要

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.

描述：（改编自申请人的摘要）血小板中的转导途径可能有助于我们理解血小板在巨核细胞中的这种研究可以提供关于调节它们的生长和成熟。亚伯拉罕博士发现了一种人巨核细胞胞质酪氨酸激酶相关粘着斑酪氨酸激酶（RAFTK）。后者属于焦点粘附激酶（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也代表了一个共同的连接整联蛋白和促有丝分裂肽的作用。人和鼠 RAFTK CDNAS已被克隆和表征。氨基酸序列也表明RAFTK类似于FAK，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将在纯化和鉴定，并研究其在信号转导中的作用。的 RAFTK的功能结构域将被定位，关系决定。将辨别RAFTK表达是否是造血增殖和分化所需的。在具体目标I中，将对RAFTK进行生物化学表征，免疫化学 RAFTK的cDNA编码结构域将在大肠杆菌中表达。 E.杆菌将纯化表达的重组蛋白。的CDNA 其编码完整的RAFTK将在杆状病毒系统中表达。的将研究重组蛋白作为激酶的性质，将产生针对重组RAFTK的单克隆抗体。抗体将开发识别RAFTK蛋白中不同结构域的方法。将描述RAFTK的不同调控域的试剂将被用于RAFTK蛋白及其上游和下游基板。在具体目标二中， RAFTK蛋白在血小板活化和凋亡信号转导中的作用将描绘巨核细胞增殖。这将涉及测量巨核细胞-骨髓成纤维细胞粘附的实验和研究巨核细胞和内皮细胞之间的相互作用。蛋白与RAFTK相互作用的蛋白质将在这些实验中鉴定。在具体目标III将绘制RAFTk的功能结构域，确定蛋白质的结构与功能关系。序列将确定RAFTK功能和本地化所需的组件。将分析主要酪氨酸磷酸化位点。富脯氨酸氨基 RAFTK中的酸序列将根据功能性重要性在具体目标四中，将确定RAFTK是否表达是造血增殖和分化所必需的。 RAFTK的基因位点将在胚胎干细胞中被破坏，同源重组不能表达RAFTK的小鼠将生成。造血缺陷小鼠的表型发展将受到审查。因此，胚胎中的靶向实验干细胞将被设计用于测试RAFTK在发育中的功能，以及造血对RAFTK的需求。