title: In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions.
abstract: 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/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-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 beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-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 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.
authors.affiliation: Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Vic. 3052, Australia.
authors.forename: Bruno
authors.initials: B
authors.lastname: Catimel
authors.forename: Meredith
authors.initials: M
authors.lastname: Layton
authors.forename: Nicole
authors.initials: N
authors.lastname: Church
authors.forename: Janine
authors.initials: J
authors.lastname: Ross
authors.forename: Melanie
authors.initials: M
authors.lastname: Condron
authors.forename: Maree
authors.initials: M
authors.lastname: Faux
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Simpson
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D015820:Cadherins / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D015820:Cadherins; D011994:Recombinant Proteins; D051176:beta Catenin