Membrane Binding of the N-Terminal Ubiquitin-Like Domain of kindlin-2 Is Crucial for Its Regulation of Integrin Activation

Abstract

SUMMARY Kindlin-2 belongs to an emerging class of regulators for heterodimeric (α/β) integrin adhesion receptors. By binding to integrin β cytoplasmic tail via its C-terminal FERM-like domain, kindlin-2 promotes integrin activation. Intriguingly, this activation process depends on the N-terminus of kindlin-2 (K2-N) which precedes the FERM domain. The molecular function of K2-N is unclear. We present the solution structure of K2-N, which displays a ubiquitin fold similar to that observed in kindlin-1. Using chemical shift mapping and mutagenesis, we found that K2-N contains a conserved positively charged surface that binds to membrane enriched with negatively charged phosphatidylinositol-(4,5)-bisphosphate. We show that while wild-type kindlin-2 is capable of promoting integrin activation, such ability is significantly reduced for its membrane-binding defective mutant. These data suggest a membrane-binding function of the ubiquitin-like domain of kindlin-2, which is likely common for all kindlins to promote their localization to the plasma membrane and control integrin activation.

A Conserved Lipid-binding Loop in the Kindlin FERM F1 Domain Is Required for Kindlin-mediated αIIbβ3 Integrin Coactivation

“…A PH domain-mediated kindlin-2-lipid interaction was previously implicated in integrin activation and while our work was in revision additional papers confirmed the importance of membrane binding via the PH and F0 domains for integrin activation (63,64). Our data extend this to show that additional membrane-targeting sites are also required, and specifically that a polylysine motif in the F1 loop is needed.…”

“…We further show that the F1 loop and its membrane binding activity are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 or kindlin-2 and the talin head in ␣IIb␤3 integrin activation, but not for kindlin binding to integrin ␤ tails. These studies highlight the structural and functional similarities between the talin head and kindlins (23,24) and, together with reports revealing a role for the kindlin F0 and PH domains in ␣IIb␤3 activation (38,51,63), indicate that multiple lipid interactions contribute to the ability of kindlins to activate integrins.…”

“…However, additional sites in kindlin are also involved as deletion of N-terminal kindlin domains ablates the ability of kindlin to cooperate with talin during ␣IIb␤3 activation (38,51,63). N-terminal domains in the talin head also contribute to talin-mediated integrin activation (36) and we recently showed the importance of a membrane-binding loop within the talin F1 domain for integrin activation (24).…”

Kindlin supports platelet integrin αIIbβ3 activation by interacting with paxillin

“…A conserved loop in the F 0 subdomain of kindlin-1 is involved in mediating the interaction with PXN and supporting integrin αIIbβ3 activation The F 0 subdomains of kindlins adopt a ubiquitin-like fold (Goult et al, 2009;Li et al, 2017;Perera et al, 2011). To identify the PXN binding sites in kindlin-1 (Fig.…”

“…3A), which is distal from the known binding sites for other molecules such as F-actin and phospholipids (Bledzka et al, 2016;Perera et al, 2011), implying that the M3 region in kindlin-1 provides a unique docking site for PXN. In addition, when we mutated each of the residues in the M3 (Q 54 DWSD) of kindlin-1, we found that each of the conserved residues (DWSD) exhibited significant influence on integrin αIIbβ3 activation, except for the nonconserved Q 54 (Fig.…”