De Silva Chamika, Kelch Brian A, McCollum Dannel
Department of Biochemistry & Molecular Biotechnology, UMass Chan Medical School, Worcester, MA, 01605.
bioRxiv. 2025 Aug 27:2025.08.26.672419. doi: 10.1101/2025.08.26.672419.
The LIM domain protein LIMD1 is a critical regulator of the Hippo signaling pathway, acting to sequester the kinases LATS1/2 to adherens junctions (AJs) in response to mechanical strain. Here, we identify the molecular basis for LIMD1 binding and recruitment of LATS1/2 to AJs. We show that while the LIM domains of LIMD1 are sufficient for AJ localization and binding to LATS1/2, recruitment of LATS1 to AJ requires both the intrinsically disordered region (IDR) in the N-terminus as well as the LIM domains. We further dissected the LIM domains and found that LIM1 and LIM2, but not LIM3, are necessary for LATS1 AJ localization. Point mutations that disrupt strain sensitivity in either the first or second LIM domain disrupt both binding and recruitment of LATS1/2 to AJs. Mechanistically, LIMD1 binds LATS1/2 through a conserved linear motif, the LATS-LATCH, which we identified by AlphaFold modeling and confirmed by biochemical and localization assays. The LATS-LATCH is both necessary and sufficient for strain-dependent recruitment of LATS1/2 to AJs. Mutation of predicted contact residues within the LATS-LATCH both disrupts its binding to LIMD1 and localization to AJs. These findings define a bipartite mechanism for LIMD1-dependent recruitment of LATS1/2 involving LIM domain-LATCH interactions and N-terminal IDR functions, providing insight into how mechanical signals are transduced through the Hippo pathway.
LIM结构域蛋白LIMD1是Hippo信号通路的关键调节因子，可响应机械应变将激酶LATS1/2隔离至黏附连接（AJs）。在此，我们确定了LIMD1将LATS1/2结合并募集至AJs的分子基础。我们发现，虽然LIMD1的LIM结构域足以实现AJ定位并与LATS1/2结合，但将LATS1募集至AJ既需要N端的内在无序区域（IDR），也需要LIM结构域。我们进一步剖析了LIM结构域，发现LIM1和LIM2而非LIM3是LATS1在AJ定位所必需的。破坏第一个或第二个LIM结构域中应变敏感性的点突变会破坏LATS1/2与AJs的结合和募集。从机制上讲，LIMD1通过一个保守的线性基序LATS-LATCH与LATS1/2结合，我们通过AlphaFold建模确定了该基序，并通过生化和定位分析进行了验证。LATS-LATCH对于依赖应变的LATS1/2募集至AJs既是必需的也是充分的。LATS-LATCH内预测的接触残基发生突变会破坏其与LIMD1的结合以及在AJs处的定位。这些发现定义了一种由LIMD1介导的LATS1/2募集的双部分机制，涉及LIM结构域-LATCH相互作用和N端IDR功能，为机械信号如何通过Hippo通路转导提供了见解。
