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Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates

Sörensson, Carolin LU ; Lenman, Marit; Veide-Vilg, Jenny; Schopper, Simone LU ; Ljungdahl, Thomas; Grotli, Morten; Tamas, Markus J.; Peck, Scott C. and Andreasson, Erik (2012) In Biochemical Journal 446. p.271-278
Abstract
MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic... (More)
MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Arabidopsis, AtIg13390.1, AtIg80180.1, At2g14850.1, At3g16770.1, mitogen-activated protein kinase substrate (MAPK substrate), random, positional scanning synthetic combinatorial peptide library, stoma
in
Biochemical Journal
volume
446
pages
271 - 278
publisher
Portland Press Limited
external identifiers
  • wos:000308767500011
  • scopus:84865190361
ISSN
0264-6021
DOI
10.1042/BJ20111809
language
English
LU publication?
yes
id
67744ba0-02ea-4f90-afdd-f8011b07fd62 (old id 3187996)
date added to LUP
2012-12-06 12:05:53
date last changed
2017-10-22 04:22:02
@article{67744ba0-02ea-4f90-afdd-f8011b07fd62,
  abstract     = {MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.},
  author       = {Sörensson, Carolin and Lenman, Marit and Veide-Vilg, Jenny and Schopper, Simone and Ljungdahl, Thomas and Grotli, Morten and Tamas, Markus J. and Peck, Scott C. and Andreasson, Erik},
  issn         = {0264-6021},
  keyword      = {Arabidopsis,AtIg13390.1,AtIg80180.1,At2g14850.1,At3g16770.1,mitogen-activated protein kinase substrate (MAPK substrate),random,positional scanning synthetic combinatorial peptide library,stoma},
  language     = {eng},
  pages        = {271--278},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates},
  url          = {http://dx.doi.org/10.1042/BJ20111809},
  volume       = {446},
  year         = {2012},
}