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.
Highlights
Integrins are heterodimeric transmembrane proteins that mediate cell adhesion to extracellular matrix proteins
We detected binding of the two anti-β1-pTpT antibodies to chimeric β3/β5-pTpT peptides and an unrelated peptide with a bi-phosphorylated double-threonine motif derived from the cytosolic scaffolding protein Wwc2 (Hoffken et al, 2021) (Fig 1D and E), indicating that the anti-β1-pTpT antibodies are not specific for β1-pT788/pT789 and react with phosphorylated bi-threonine motifs in β1 integrin-unrelated proteins
We analyzed β1-pT788/pT789 levels with the anti-β1-pTpT antibodies in lysates from spread interphase and mitotic mouse fibroblasts seeded on fibronectin (FN)
Summary
Integrins are heterodimeric transmembrane proteins that mediate cell adhesion to extracellular matrix proteins. Integrin activation is induced and/or maintained by the binding of the FERM (protein 4.1, ezrin, radixin, and moesin) domain-containing adaptor proteins talin and kindlin (Cluzel et al, 2005; Han et al, 2006; Moser et al, 2008; Ye et al, 2013; Theodosiou et al, 2016; Bottcher et al, 2017) to conserved NxxY motifs and adjacent residues in β-integrin cytoplasmic domains (β-tail). The phosphorylation of the tyrosine residues in the NxxY motifs of the β1-tail is mediated by Src (Hirst et al, 1986; Johansson et al, 1994) resulting in reduced talin and kindlin binding (Anthis et al, 2009; Meves et al, 2011). In the case of the β1-tail threonine phosphorylation, both the identity of the kinase as well as the functional consequences of the phosphorylation event are less investigated and less clear
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