ILK binding to β1 integrin is indirect and mediated by kindlin-2.

Integrin-linked kinase (ILK) and kindlin-2 (K2) are key components of focal adhesions (FAs) that regulate cell-matrix adhesion and integrin signaling. Both proteins directly bind each other, but how they influence each other's localization to FAs and binding to integrins remains a subject of ongoing debate. Here, we establish a sensitive workflow to study protein-protein interactions in cells by combining methods from biochemistry, cell biology, and superresolution microscopy. Together with an analytical framework, this approach allowed us to distinguish direct from indirect molecular interactions and construct detailed interaction networks. Disrupting the ILK-K2 interaction reduced ILK localization to FAs and compromised integrin function, whereas K2 recruitment was unaffected. Our interdisciplinary approach also revealed that ILK does not directly bind β1-integrin cytosolic domains in vitro and in cells. Instead, ILK was recruited to integrins exclusively through a K2-dependent mechanism, primarily via K2 bridging ILK and β1 integrins. These findings define the hierarchical relationship between ILK and K2 in FAs and highlight the essential role of K2-mediated ILK recruitment for integrin adhesion and signaling.