title: Characterization of a novel calcium/calmodulin-dependent protein kinase from tobacco.
abstract: 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 Km of 44.5 microm and a Vmax of 416.2 nm min(-1) mg(-1). Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 22.1 microm and a Vmax 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 (Kd = 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.
authors.affiliation: Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Shuping
authors.initials: S
authors.lastname: Liang
authors.forename: Russell L
authors.initials: RL
authors.lastname: Jones
authors.forename: Ying-Tang
authors.initials: YT
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D010446:Peptide Fragments; D010940:Plant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases