Department of Chemistry, University of California, Berkeley, Berkeley, United States.
California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States.
Elife. 2023 Apr 26;12:e82676. doi: 10.7554/eLife.82676.
The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a 'Src module', an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP, or in solution by inositol hexakisphosphate (IP) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues - thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.
Tec 家族激酶 Btk 包含一个脂质结合的 Pleckstrin 同源和 Tec 同源（PH-TH）模块，通过富含脯氨酸的接头与“Src 模块”连接，Src 家族激酶和 Abl 中也发现了该模块。我们之前表明，Btk 通过 PH-TH 二聚化激活，这种二聚化在膜上由磷脂酰肌醇磷酸盐 PIP 触发，或在溶液中由肌醇六磷酸（IP）触发（Wang 等人，2015 年，https://doi.org/10.7554/eLife.06074）。我们现在报告说，普遍存在的衔接蛋白生长因子受体结合蛋白 2（Grb2）与 PIP 结合的 Btk 结合，并大大增加其在膜上的活性。使用在支持脂双层上的重组，我们发现 Grb2 可以通过与 Btk 中的富含脯氨酸的接头相互作用被募集到膜结合的 Btk。这种相互作用需要完整的 Grb2，包含两个 SH3 结构域和 SH2 结构域，但不需要 SH2 结构域能够结合磷酸化的酪氨酸残基 - 因此，与 Btk 结合的 Grb2 可以自由通过 SH2 结构域与支架蛋白相互作用。我们表明，Grb2-Btk 相互作用将 Btk 募集到重组膜中的支架介导的信号簇中。我们的发现表明，Btk 的 PIP 介导的二聚化不完全激活 Btk，并且 Grb2 通过释放 Btk 使其在膜上处于自动抑制状态。
