In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-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/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.
Catimel
Bruno
B
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Vic. 3052, Australia.
Layton
Meredith
M
Church
Nicole
N
Ross
Janine
J
Condron
Melanie
M
Faux
Maree
M
Simpson
Richard J
RJ
Burgess
Antony W
AW
Nice
Edouard C
EC
D015374:Biosensing Techniques / Q000295:instrumentation*; D015820:Cadherins / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins; D051176:beta Catenin / Q000378:metabolism*
D015820:Cadherins; D011994:Recombinant Proteins; D051176:beta Catenin