Abstract
Integrin-linked kinase (ILK) is a distinct intracellular adaptor essential for integrin-mediated cell-extracellular matrix adhesion, cell spreading, and migration. Acting as a major docking platform in focal adhesions, ILK engages many proteins to dynamically link integrins with the cytoskeleton, but the underlying mechanism remains elusive. Here, we have characterized the interaction of ILK with kindlin-2, a key regulator for integrin bidirectional signaling. We show that human kindlin-2 binds to human ILK with high affinity. Using systematic mapping approaches, we have identified a major ILK binding site involving a 20-residue fragment (residues 339-358) in kindlin-2. NMR-based analysis reveals a helical conformation of this fragment that utilizes its leucine-rich surface to recognize the ILK pseudokinase domain in a mode that is distinct from another ILK pseudokinase domain binding protein, α-parvin. Structure-based mutational experiments further demonstrate that the kindlin-2 binding to ILK is crucial for the kindlin-2 localization to focal adhesions and cell spreading (integrin outside-in signaling) but dispensable for the kindlin-2-mediated integrin activation (integrin inside-out signaling). These data define a specific mode of the kindlin-2/ILK interaction with mechanistic implications as to how it spatiotemporally mediates integrin signaling and cell adhesion.
Highlights
Mechanism of the kindlin-2/Integrin-linked kinase (ILK) interaction remains elusive
Using NMR, we further showed that 2H,15N-labeled heteronuclear single quantum coherence (HSQC) spectrum of a double kindlin-2 F2 mutant (L353A and L357A; hereinafter referred to as LL/AA) exhibited a well dispersed spectrum that is similar to the wild type but has no chemical shift perturbation and line broadening upon addition of unlabeled ILK KLD1⁄7␣-parvin calponin homology domain 2 (CH2) (Fig. 5B)
We have focused on analyzing the interaction of two major integrin regulators, kindlin-2 and ILK
Summary
Mechanism of the kindlin-2/ILK interaction remains elusive. Results: Kindlin-2 recognizes the ILK pseudokinase domain via a leucine-rich amphipathic ␣-helix, and such binding is crucial for focal adhesion assembly and cell spreading. Structurebased mutational experiments further demonstrate that the kindlin-2 binding to ILK is crucial for the kindlin-2 localization to focal adhesions and cell spreading (integrin outside-in signaling) but dispensable for the kindlin-2-mediated integrin activation (integrin inside-out signaling). Structure-based mutational experiments reveal that kindlin-2 binding to ILK is crucial for the kindlin-2 localization to FAs and cell spreading (integrin outside-in signaling) but dispensable for the kindlin2-mediated integrin activation (integrin inside-out signaling). These data provide important structural insights into the kindlin-2/ILK interaction and suggest a spatiotemporal pathway wherein kindlin-2 co-localizes with ILK at FAs after triggering integrin activation to dynamically regulate FA assembly and cell adhesion
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