Site-specific phosphorylation regulates the functions of kindlin-3 in a variety of cells.

Studies of isolated cells, mice, and humans have demonstrated the vital role of the FERM domain protein kindlin-3 in integrin activation in certain hematopoietic and non-hematopoietic cells, consequent to binding to integrin β-subunits. To explore regulatory mechanisms, we developed a monoclonal antibody that selectively recognizes the phosphorylated form of Ser484 (pS484) in kindlin-3. Activation of platelets, HEL megakaryocytic-like cells and BT549 breast cancer cells led to enhanced expression of pS484 as assessed by immunofluorescence or Western blotting. In platelets, pS484 rose rapidly and transiently upon stimulation. When a mutant form of kindlin-3, T482S484/AA kindlin-3, was transduced into mouse megakaryocytes, it failed to support activation of integrin αIIbβ3, whereas wild-type kindlin-3 did. In MDA-MB231 breast cancer cells, expression of T482S484/AA kindlin-3 suppressed cell spreading, migration, invasion, and VEGF production. Wild-type kindlin-3 expressing cells markedly increased tumor growth in vivo, whereas T482S484/AA kindlin-3 significantly blunted tumor progression. Thus, our data establish that a unique phosphorylation event in kindlin-3 regulates its cellular functions.