Tyrosine Phosphorylation of Integrin 3 Regulates Kindlin-2 Binding and Integrin Activation

Department of Molecular Cardiology, Joseph J Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2010; 285(40):30370-4. DOI: 10.1074/jbc.C110.134247
Source: PubMed


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.

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    • "Previous studies have shown that integrin β1-Y783 and -Y795 are parts of conserved NPxY motifs essential for recruiting talin and kindlin, which in turn facilitates coupling of integrin β1 to the actin cytoskeleton and maintains integrins in an active signaling state [33]. It was reported that v-Src could phosphorylate integrin β1 tails on Y783 and Y795 [34,35], and that phosphorylated Y783 and Y795 could block talin and kindlin’s binding with integrin β1, respectively [36,37]. These were supported by the study that v-Src expression in fibroblasts decreases integrin β1–dependent adhesion, focal adhesion formation, cytoskeletal organization, fibronectin assembly, migration, and chemotaxis [34,38]. "
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    ABSTRACT: Background Phosphatase of regenerating liver-3 (PRL-3 or PTP4A3) has been implicated in controlling cancer cell proliferation, motility, metastasis, and angiogenesis. Deregulated expression of PRL-3 is highly correlated with cancer progression and predicts poor survival. Although PRL-3 was categorized as a tyrosine phosphatase, its cellular substrates remain largely unknown. Results We demonstrated that PRL-3 interacts with integrin β1 in cancer cells. Recombinant PRL-3 associates with the intracellular domain of integrin β1 in vitro. Silencing of integrin α1 enhances PRL-3-integrin β1 interaction. Furthermore, PRL-3 diminishes tyrosine phosphorylation of integrin β1 in vitro and in vivo. With site-specific anti-phosphotyrosine antibodies against residues in the intracellular domain of integrin β1, tyrosine-783, but not tyrosine-795, is shown to be dephosphorylated by PRL-3 in a catalytic activity-dependant manner. Phosphorylation of Y783 is potentiated by ablation of PRL-3 or by treatment with a chemical inhibitor of PRL-3. Conversely, depletion of integrin α1 decreases the phosphorylation of this site. Conclusions Our results revealed a direct interaction between PRL-3 and integrin β1 and characterized Y783 of integrin β1 as a bona fide substrate of PRL-3, which is negatively regulated by integrin α1.
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    • "In contrast, we saw no statistically significant effect. Our results are consistent with previous findings that THD can activate αIIbβ3 mutants with markedly reduced affinity for kindlins [17], [41]. Our results also agree with the observation that THD alone is sufficient to activate integrins reconstituted in lipid bilayers [17]. "
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    ABSTRACT: Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.
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