Tyrosine phosphorylation of integrin beta3 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 NITY(759) motif in the β(3) 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 β(3) CT interacted well with kindlin-2, whereas the Tyr(759)-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 β(3) CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.

FIGURE 1.

Phosphomimetic mutation of the NxxY⁷⁵⁹ motif in the β₃ CT perturbs kindlin-2 binding and suppresses kindlin-2-mediated integrin α_IIbβ₃ activation. A, kindlin-2 and talin-H can interact with the integrin β₃ CT. The kindlin-2-binding site is localized in the membrane-distal NxxY motif, NITY⁷⁵⁹, but talin-H binding depends on the membrane-proximal NxxY motif, NPLY⁷⁴⁷. B, GST-fused β₃ CT, with or without the indicated mutations, was used in pulldown assays to precipitate kindlin-2 from cell lysates. The bound kindlin-2 was measured by Western blotting. IB, immunoblot. C, a representative FACS histogram developed with the activation-specific PAC1 mAb is shown together with the median fluorescence intensities (MFI) demonstrating that total α_IIbβ₃ expression, measured with a mAb unaffected by the activation status of the receptor, is unaffected. D, wild-type α_IIbβ₃ and its mutants were coexpressed with talin-H and kindlin-2 or their empty vectors in CHO cells by transient transfection, and the extent of α_IIbβ₃ activation was analyzed by PAC1 binding of the DsRed and EGFP double-positive cells, correcting for any differences in total α_IIbβ₃ expression. Results are means ± S.D. (n = 3). **, p < 0.01.

FIGURE 2.

Phosphorylation of the NxxY⁷⁵⁹ motif in the β₃ CT peptides disables its ability to block interaction of kindlin-2 and the β₃ CT. A, the amino acid sequences of the β₃ CT C-terminal peptide containing the NITY⁷⁵⁹ motif and a modified peptide with Tyr⁷⁵⁹ phosphorylation (β3C-pep-phos) are shown. B, kindlin-2 and β₃ CT interaction was evaluated by pulldown assays in the presence of the indicated peptides. IB, immunoblot. C, kindlin-2 or a mixture of kindlin-2 and the indicated peptide at a 1:100 molar ratio was passed over an SA5 biosensor chip coated with biotinylated β₃ CT peptides, and the binding curves were recorded over time. To block nonspecific binding of the peptide to the chips, 0.1% BSA was included in the running buffer.

FIGURE 3.

Phosphorylation of the NxxY⁷⁵⁹ motif in the β₃ CT peptides dampens direct binding to kindlin-2. Biotinylated β₃ CT peptides with or without modification at Tyr⁷⁵⁹ are displayed (A). Phos, phosphorylated. The WT biotin-β₃ peptide (B), its phosphorylated form (C), and other mutants (D) were immobilized on the streptavidin sensor chips. Various concentrations of kindlin-2 protein were injected over the chips in HEPES buffer (0.1% BSA) at a flow rate of 25 μl/min. The binding signals were recorded, and the results are expressed in RU. The RU values of the chips prior to introduction of kindlin-2 were similar (∼200 RU), indicating that the coating densities of the peptides were similar.

FIGURE 4.

Phosphorylation of Tyr⁷⁹⁵ in the β₁ peptide inhibits kindlin-2 binding. A, the amino acid sequence of the β₁ CT is shown. B, the inhibitory effects of the indicated β₁ peptides on kindlin-2 and β₃ CT interaction were evaluated in pulldown assays, and kindlin-2 binding was evaluated by Western blotting. IB, immunoblot; β1C-pep-phos, Tyr⁷⁹⁵-phosphorylated β₁ CT C-terminal peptide.