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Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins

O'Connell, David; Bauer, Mikael LU ; O'Brien, John; Johnson, Winifred M; Divizio, Catherine A; O'Kane, Sara L; Berggård, Tord LU ; Merino, Alejandro; Akerfeldt, Karin and Linse, Sara LU , et al. (2010) In Molecular & Cellular Proteomics 9(6). p.1118-1132
Abstract
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-labelled 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) = 1 muM) 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... (More)
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-labelled 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) = 1 muM) 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 (koff = 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 have developed a microarray of the identified target proteins with which we can characterise 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), CaM kinase-like vesicle-associated protein (302-316), EF-hand domain family member A2 (202-216) and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (400-415). (Less)
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Contribution to journal
publication status
published
subject
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Molecular & Cellular Proteomics
volume
9
issue
6
pages
1118 - 1132
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000279396900006
  • pmid:20068228
  • scopus:77953141357
ISSN
1535-9484
DOI
10.1074/mcp.M900324-MCP200
language
English
LU publication?
yes
id
4703c3a7-9b3c-4f2f-bc97-a1c1330a1cec (old id 1541151)
date added to LUP
2010-02-02 14:38:25
date last changed
2018-05-29 10:28:29
@article{4703c3a7-9b3c-4f2f-bc97-a1c1330a1cec,
  abstract     = {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-labelled 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) = 1 muM) 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 (koff = 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 have developed a microarray of the identified target proteins with which we can characterise 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), CaM kinase-like vesicle-associated protein (302-316), EF-hand domain family member A2 (202-216) and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (400-415).},
  author       = {O'Connell, David and Bauer, Mikael and O'Brien, John and Johnson, Winifred M and Divizio, Catherine A and O'Kane, Sara L and Berggård, Tord and Merino, Alejandro and Akerfeldt, Karin and Linse, Sara and Cahill, Dolores J},
  issn         = {1535-9484},
  language     = {eng},
  number       = {6},
  pages        = {1118--1132},
  publisher    = {American Society for Biochemistry and Molecular Biology},
  series       = {Molecular & Cellular Proteomics},
  title        = {Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins},
  url          = {http://dx.doi.org/10.1074/mcp.M900324-MCP200},
  volume       = {9},
  year         = {2010},
}