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Published on July 9, 2004; 10.1104/pp.104.041970

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Received March 2, 2004
Returned for revision March 22, 2004
Accepted April 7, 2004

Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase from Tobacco

Li Ma , Shuping Liang , Russell L. Jones , and Ying-Tang Lu *

Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720

* Corresponding author; email: yingtlu{at}whu.edu.cn.

A cDNA encoding a calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CaMK) from tobacco (Nicotiana tabacum), NtCaMK1, was isolated by protein-protein interaction-based screening of a cDNA expression library using 35S-labeled CaM as a probe. The genomic sequence is about 24.6 kb, with 21 exons, and the full-length cDNA is 4.8 kb, with an open reading frame for NtCaMK1 consisting of 1,415 amino acid residues. NtCaMK1 has all 11 subdomains of a kinase catalytic domain, lacks EF hands for Ca2+-binding, and is structurally similar to other CaMKs in mammal systems. Biochemical analyses have identified NtCaMK1 as a Ca2+/CaMK since NtCaMK1 phosphorylated itself and histone IIIs as substrate only in the presence of Ca2+/CaM with a K m of 44.5 µ M and a V max of 416.2 nM min-1 mg-1. Kinetic analysis showed that the kinase not previously autophosphorylated had a K m for the synthetic peptide syntide-2 of 22.1 µM and a V max of 644.1 nM min-1 mg-1 when assayed in the presence of Ca2+/CaM. Once the autophosphorylation of NtCaMK1 was initiated, the phosphorylated form displayed Ca2+/CaM-independent behavior, as many other CaMKs do. Analysis of the CaM-binding domain (CaMBD) in NtCaMK1 with truncated and site-directed mutated forms defined a stretch of 20 amino acid residues at positions 913 to 932 as the CaMBD with high CaM affinity (K d = 5 nM). This CaMBD was classified as a 1-8-14 motif. The activation of NtCaMK1 was differentially regulated by three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13). While NtCaM1 and NtCaM13 activated NtCaMK1 effectively, NtCaM3 did not activate the kinase.




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