Tobacco calcium-dependent protein kinases are differentially phosphorylated in vivo as part of a kinase cascade that regulates stress response.
In vivo phosphorylation sites of the tobacco calcium-dependent protein kinases NtCDPK2 and NtCDPK3 were determined in response to biotic or abiotic stress. Stress-inducible phosphorylation was exclusively located in the variable N termini, where both kinases were phosphorylated differentially despite 91% overall sequence identity. In NtCDPK2, serine 40 and threonine 65 were phosphorylated within 2 min after stress. Whereas Thr(65) is subjected to intra-molecular in vivo autophosphorylation, Ser(40) represents a target for a regulatory upstream protein kinase, and correct NtCDPK2 membrane localization is required for Ser(40) phosphorylation. NtCDPK3 is phosphorylated at least at two sites in the N terminus by upstream kinase(s) upon stress stimulus, first at Ser(54), a site not present in NtCDPK2, and also at a second undetermined site not identical to Ser(40). Domain swap experiments established that differential phosphorylation of both kinases is exclusively determined by the respective N termini. A cell death-inducing response was only observed upon expression of a truncated variant lacking the junction and calcium-binding domain of NtCDPK2 (VK2). This response required protein kinase activity and was reduced when subcellular membrane localization was disturbed by a mutation in the myristoylation and palmitoylation site. Our data indicate that CDPKs are integrated in stress-dependent protein kinase signaling cascades, and regulation of CDPK function in response to in vivo stimulation is dependent on its membrane localization.
Witte
Claus-Peter
CP
Department of Plant Biochemistry, Institute for Biology, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany.
Keinath
Nana
N
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50935 Cologne, Germany.
Dubiella
Ullrich
U
Department of Plant Biochemistry, Institute for Biology, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany.
Demoulière
Raphael
R
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50935 Cologne, Germany.
Seal
Anindita
A
Department of Biotechnology, West Bengal University of Technology BF-142, Sector I, Salt Lake, Calcutta 700064, India.
Romeis
Tina
T
Department of Plant Biochemistry, Institute for Biology, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany. Electronic address: romeis@zedat.fu-berlin.de.
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D002118:Calcium / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014026:Nicotiana / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases