Differences in self-association between kindlin-2 and kindlin-3 are associated with differential integrin binding

Yasmin A Kadry,Eesha M Maisuria,Clotilde Huet-Calderwood,David A Calderwood

doi:10.1074/jbc.ra120.013618

Abstract

The integrin family of transmembrane adhesion receptors coordinates complex signaling networks that control the ability of cells to sense and communicate with the extracellular environment. Kindlin proteins are a central cytoplasmic component of these networks, directly binding integrin cytoplasmic domains and mediating interactions with cytoskeletal and signaling proteins. The physiological importance of kindlins is well established, but how the scaffolding functions of kindlins are regulated at the molecular level is still unclear. Here, using a combination of GFP nanotrap association assays, pulldown and integrin-binding assays, and live-cell imaging, we demonstrate that full-length kindlins can oligomerize (self-associate) in mammalian cells, and we propose that this self-association inhibits integrin binding and kindlin localization to focal adhesions. We show that both kindlin-2 and kindlin-3 can self-associate and that kindlin-3 self-association is more robust. Using chimeric mapping, we demonstrate that the F2PH and F3 subdomains are important for kindlin self-association. Through comparative sequence analysis of kindlin-2 and kindlin-3, we identify kindlin-3 point mutations that decrease self-association and enhance integrin binding, affording mutant kindlin-3 the ability to localize to focal adhesions. Our results support the notion that kindlin self-association negatively regulates integrin binding.

Highlights

The phenotypes of kindlin knockout or depletion in mouse, Drosophila, and Caenorhabditis elegans clearly establish the general importance of kindlins for integrin function [1,2,3,4,5,6]
We report that full-length kindlin-2 and kindlin-3 can each self-associate in mammalian cells but that kindlin-3 self-associates to a greater extent than kindlin-2
The crystal structure of kindlin-2 lacking the pleckstrin homology (PH) domain and a portion of the F1 subdomain suggested that kindlin-2DPHDF1loop dimerizes [14], it was unclear whether full-length kindlin-2 or the related kindlin-3 could self-associate in mammalian cells

Summary

Discussion

Kindlins are essential for normal integrin-mediated cell adhesion and signaling [1, 18]. Mutations in the F3 subdomain of kindlin-3 that impair self-association of kindlin-3 enhance binding to b1 integrin cytoplasmic tails and permit localization of kindlin-3 to focal adhesions, suggesting that kindlin dimerization or oligomerization regulates integrin binding and localization. We find that kindlin-3 self-associates to a greater extent than kindlin-2, raising the possibility that kindlin-self-association is inversely correlated with integrin binding and focal adhesion localization. It is not immediately clear how to rationalize the increased binding of the compound kindlin-3 mutant to integrin tails, raising the possibility that the effect is indirect Consistent with this idea, we found that kindlin-3 F3mutx is impaired in self-association. This suggests that it is the impairment in self-association that enhances integrin binding and increases targeting to focal adhesions of the compound kindlin-3 mutant. In light of our findings that kindlin self-association inversely correlates with integrin binding, it will be important to test the consequences of selectively modulating kindlin-3 selfassociation on cell adhesion and spreading and integrin activation and signaling in these cells

Experimental procedures

Cell culture and transfection

TIRF microscopy

Integrin binding assays