In situ phosphorylation of immobilized receptors on biosensor surfaces: Application to E-cadherin/β-catenin interactions

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein–protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/β-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to β-catenin and consequently the strength of cell–cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of β-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to β-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 μM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of β-catenin binding by src phosphorylation.