Characterization of a novel calcium/calmodulin-dependent protein kinase from tobacco.
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.
Ma
Li
L
Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Liang
Shuping
S
Jones
Russell L
RL
Lu
Ying-Tang
YT
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*
D018076:DNA, Complementary; D010446:Peptide Fragments; D010940:Plant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases