Tyrosine phosphorylation of integrin β3 regulates kindlin-2 binding and integrin activation

Abstract

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY759 motif in the β3 CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β3 CT interacted well with kindlin-2, whereas the Tyr759-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β3 CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.