Site specific phosphorylation of kindlin-3 regulates its capacity to control cellular responses mediated by integrin {alpha}IIb{beta}3 [Signal Transduction]

January 21st, 2015 by Bialkowska, K., Byzova, T. V., Plow, E. F.

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins by their cytoplasmic tails. Kindlins are integrin cytoplasmic tail (CT) binding partners, are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin αIIbβ3, to investigate whether phosphorylation of kindlin-3 regulate integrin activation. When HEL cells were stimulated with thrombopoietin (TPO) or phorbol 12-mirystate 13-acetate (PMA), αIIbβ3 became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β3 integrin and kindlin-3 in focal adhesions, and enhanced β3 integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. T482 or S484 was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482S484/AA kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared to wild-type kindlin-3. A membrane-permeable peptide containing residues 476-485 of kindlin-3 was introduced into HEL cells and platelets, adhesion and spreading of both cell types was blunted compared to a scrambled control peptide. These data identify a role kindlin-3 phosphorylation in integrin β3 activation and provide a basis for functional differences between kindlin-3 and other two kindlin paralogs.
  • Posted in Journal of Biological Chemistry, Publications
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