New insights into the phosphorylation of the threonine motif of the ß1 integrin cytoplasmic domain.

ABSTRACT

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the ß integrin cytosolic domain (ß-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the ß1-tail (ß1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against ß1-pT788/pT789 integrin do not detect specific ß1-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 ß1-TT788/789DD integrin failed to activate ß1 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 ß1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in ß1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.