Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca(2+)-dependent protein kinase is important for substrate recognition.
Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca(2+)-dependent protein kinases (CDPKs) play central roles in Ca(2+) signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways.
Ito
Takeshi
T
Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Japan.
Nakata
Masaru
M
Fukazawa
Jutarou
J
Ishida
Sarahmi
S
Takahashi
Yohsuke
Y
D019943:Amino Acid Substitution / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000235:genetics; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D029681:Arabidopsis Proteins; D001120:Arginine; D050976:Basic-Leucine Zipper Transcription Factors; D002118:Calcium; D002135:Calcium-Binding Proteins; D010940:Plant Proteins; D011494:Protein Kinases; D012097:Repressor Proteins; C490306:ATCDPK1 protein, Arabidopsis; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C412626:RSG protein, Nicotiana tabacum