New methods for activation of kinases and kinase circuits in living cells.

激活活细胞中激酶和激酶电路的新方法。

• 批准号: 8243734
• 负责人: ANDREI V KARGINOV
• 金额: $ 20.81万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8243734
• 关键词: Affect; Binding; Biological; Biological Models; Biological Process; CCI-779; Calcineurin; Catalytic Domain; Cell Surface Extensions; Cells; Dependence; Development; Dimerization; Disease; Dose; Engineering; Event; FK506; Family member; Focal Adhesion Kinase 1; Focal Adhesions; Heterodimerization; Human; Kinetics; Lead; Life; Light; Location; MAPK14 gene; Mediating; Membrane; Methods; Organ; Outcome; Pathway interactions; Phenotype; Phosphotransferases; Positioning Attribute; Protein Engineering; Protein Kinase; Reagent; Regulation; Resolution; Role; Scientist; Signal Pathway; Sirolimus; Specificity; System; Tacrolimus Binding Proteins; Techniques; Technology; Testing; Time; Work; analog; base; cell behavior; cell motility; inhibitor/antagonist; metaplastic cell transformation; mutant; novel strategies; protein complex; src-Family Kinases; tool

DESCRIPTION (provided by applicant): It remains difficult to manipulate protein kinase activity with precise timing and localization in living cells. Furthermore, targeted manipulation of kinase activity only in selected protein complexes is currently impossible for the majority of biological studies. We have recently developed a new generally applicable method for rapamycin-regulated (RapR) activation of kinases and successfully applied it to three kinases from two different classes, tyr and ser/thr kinases (FAK, Src, and p38). Here, we propose to employ RapR technology to develop new broadly applicable methods for selective regulation of highly homologous kinases in living cells, and targeted activation of kinases only when they are in specific protein complexes. We will also achieve light- mediated localized regulation of kinases using caged rapamycin. These methods will be applied to identify the roles of different Src family kinases. These highly homologous kinases serve as a good test of the specificity of the new approaches and will provide new capabilities to answer previously intractable questions. Localized activation will be used to probe the spatio-temporal regulation of pathways modulating cell protrusion and polarization. PUBLIC HEALTH RELEVANCE: The proposed project is focused on the development of new tools for targeted manipulation and interrogation of specific signaling pathways in live cells. In particular, the work will enable scientists to turn on kinases, an important class of regulatory molecule, in precise places and times in cells. These new approaches will allow scientists to identify biological processes critical for normal development and function of human organs as well as pathological events leading to disease.

描述（由申请人提供）：在活细胞中使用精确的时机和定位操纵蛋白激酶活性仍然很难。此外，对于大多数生物学研究，目前不可能在选定的蛋白质复合物中对激酶活性的有针对性操纵。我们最近开发了一种新的通常适用的方法，用于激酶的雷帕霉素调节（RAPR）激活，并成功地将其应用于来自Tyr和Ser/Thr激酶的两种不同类别的三个激酶（FAK，SRC和P38）。在这里，我们建议采用RAPR技术来开发新的广泛适用方法，以选择性地调节活细胞中高度同源激酶，并仅当激酶处于特定蛋白质复合物中时才靶向激活。我们还将使用笼中雷帕霉素实现激酶的光介导的局部调节。这些方法将应用于确定不同SRC家族激酶的作用。这些高度同源的激酶可以很好地测试新方法的特异性，并将提供新的能力来回答以前棘手的问题。局部激活将用于探测调节细胞突出和极化的途径的时空调节。 公共卫生相关性：拟议项目的重点是开发针对活细胞中特定信号途径的针对性操纵和询问的新工具。特别是，这项工作将使科学家能够在细胞中精确的位置和时间启用一类重要的调节分子激酶。这些新方法将使科学家能够确定对人体器官正常发育和功能至关重要的生物学过程以及导致疾病的病理事件。