Integrated protein array screening and high throughput validation of 70 novel neural calmodulin-binding proteins.
Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) < or = 1 microm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (k(off) < or = 10(-3) s(-1)) and high affinity (K(D) </= 1 mum), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474-493), calmodulin kinase-like vesicle-associated protein (residues 302-316), EF-hand domain family member A2 (residues 202-216), and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (residues 400-415).
O'Connell
David J
DJ
Translational Research Centre, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Republic of Ireland.
Bauer
Mikael C
MC
O'Brien
John
J
Johnson
Winifred M
WM
Divizio
Catherine A
CA
O'Kane
Sara L
SL
Berggård
Tord
T
Merino
Alejandro
A
Akerfeldt
Karin S
KS
Linse
Sara
S
Cahill
Dolores J
DJ
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D015203:Reproducibility of Results; D012984:Software; D020349:Surface Plasmon Resonance
D002148:Calmodulin-Binding Proteins; D010455:Peptides