Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase.
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Research Article|
March 14 2014
Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases
Kirby N. Swatek;
Kirby N. Swatek
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
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Rashaun S. Wilson;
Rashaun S. Wilson
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
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Nagib Ahsan;
Nagib Ahsan
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
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Rebecca L. Tritz;
Rebecca L. Tritz
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
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Jay J. Thelen
Jay J. Thelen
1
*Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, U.S.A.
1To whom correspondence should be addressed (email thelenj@missouri.edu).
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Publisher: Portland Press Ltd
Received:
January 04 2013
Revision Received:
December 21 2013
Accepted:
January 03 2014
Accepted Manuscript online:
January 03 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 459 (1): 15–25.
Article history
Received:
January 04 2013
Revision Received:
December 21 2013
Accepted:
January 03 2014
Accepted Manuscript online:
January 03 2014
Citation
Kirby N. Swatek, Rashaun S. Wilson, Nagib Ahsan, Rebecca L. Tritz, Jay J. Thelen; Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases. Biochem J 1 April 2014; 459 (1): 15–25. doi: https://doi.org/10.1042/BJ20130035
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