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）：在活细胞中精确定时和定位操纵蛋白激酶活性仍然很困难。此外，目前在大多数生物学研究中，仅在选定的蛋白质复合物中靶向操纵激酶活性是不可能的。我们最近开发了一种新的普遍适用的雷帕霉素调节（RapR）激活激酶的方法，并成功地将其应用于来自两种不同类型的三种激酶，tyr和ser/thr激酶（FAK， Src和p38）。在这里，我们建议利用RapR技术开发新的广泛适用的方法来选择性调节活细胞中高度同源的激酶，以及仅当激酶处于特定蛋白质复合物中时靶向激活它们。我们还将利用笼型雷帕霉素实现光介导的激酶局部调控。这些方法将用于确定不同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难以解决的问题。局部激活将用于探索调节细胞突起和极化的通路的时空调节。