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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase from Tobacco^1,[w]

Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China (L.M., S.L., Y.-T.L.); and Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (R.L.J.)

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 µM 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.

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041970.

^* Corresponding author; e-mail yingtlu{at}whu.edu.cn; fax 86–27–87666380.

Received March 2, 2004; returned for revision March 22, 2004; accepted April 7, 2004.

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