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Free keywords:
ADHESION MEASUREMENTS; INTEGRIN ACTIVATION; CELL-ADHESION; BINDING;
BETA(1)-INTEGRIN; FILAMIN; IDENTIFICATION; TYROSINES; PROTEINS; AFFINITYLife Sciences & Biomedicine - Other Topics;
Abstract:
Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the beta integrin cytosolic domain (beta-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the beta 1-tail (beta 1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against beta 1-pT788/pT789 integrin do not detect specific beta 1-pT788/ pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine resi-dues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibro-blasts and epithelial cells expressing the phospho-mimicking beta 1-TT788/789DD integrin failed to activate beta 1 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 beta 1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in beta 1-class integrins is not a major phosphory-lation site but if phosphorylated would curb integrin function.
