Abstract
Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.