Department of Biology, Stanford University, Stanford, CA, USA.
Department of Chemistry - BMC, Uppsala University, Uppsala, Sweden.
Mol Cell. 2020 Jul 16;79(2):342-358.e12. doi: 10.1016/j.molcel.2020.06.029. Epub 2020 Jul 8.
Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
短线性基序 (SLiMs) 驱动着信号转导所必需的动态蛋白质-蛋白质相互作用，但序列简并性和低结合亲和力使得它们难以识别。我们利用无偏的系统方法来发现 SLiMs，以阐明钙调神经磷酸酶 (CN)/PP2B 的调控网络，CN/PP2B 是一种识别 LxVP 和 PxIxIT 基序的 Ca 激活磷酸酶。体外全蛋白质组范围内的 CN 结合肽检测、体内 SLiM 依赖性邻近标记和基序决定因素的计算建模揭示了意想不到的 CN 相互作用因子，包括 NOTCH1，我们将其确定为 CN 的底物。出乎意料的是，CN 显示出与中心体和核孔复合物 (NPC) 蛋白的 SLiM 依赖性接近，而 Ca 信号在这些结构中很大程度上尚未被描述。CN 去磷酸化人类和酵母 NPC 蛋白，并促进核转运报告蛋白的积累，表明 NPC 受到 CN 的保守调控。这里组装的 CN 网络为研究 Ca 和 CN 信号提供了资源，并展示了实验和计算方法之间的协同作用，为研究基于 SLiMs 的网络建立了蓝图。
