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

描述（由申请人提供）：仍然难以在活细胞中精确定时和定位地操纵蛋白激酶活性。此外，目前大多数生物学研究不可能仅在选定的蛋白质复合物中靶向操纵激酶活性。我们最近开发了一种新的普遍适用的方法雷帕霉素调节（RapR）激活激酶，并成功地将其应用于三种激酶从两个不同的类，酪氨酸和丝氨酸/苏氨酸激酶（FAK，Src和p38）。在这里，我们建议采用RapR技术开发新的广泛适用的方法，用于选择性调节活细胞中高度同源的激酶，并仅当它们处于特定的蛋白质复合物中时才靶向激活激酶。我们还将利用笼状雷帕霉素实现光介导的激酶局部调节。这些方法将被应用于确定不同的Src家族激酶的作用。这些高度同源的激酶是对新方法特异性的一个很好的测试，并将提供新的能力来回答以前难以解决的问题。局部激活将用于探测调节细胞突起和极化的途径的时空调节。

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.