Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein

Abstract

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which we designated NtCDPK1. The deduced amino acid sequence of NtCDPK1 suggests that this protein contains the kinase domain at the amino terminus and the autoregulatory and calmodulin-like domains at the carboxy terminus. NtCDPK1 is highly homologous to DcCPK1, a CDPK of carrot, showing 76.5% amino acid sequence identity. NtCDPK1 transcripts are present in roots, stems and flowers, but are almost undetectable in leaves. In leaves, NtCDPK1 mRNA accumulation is stimulated by phytohormones (ABA, GA and cytokinin), Ca2+, methyl jasmonate, wounding, fungal elicitors, chitosan, and NaCl. The recombinant full-length NtCDPK1 protein is catalytically active and highly stimulated by Ca2+. A truncated recombinant NtCDPK1 which lacks the C-terminal calmodulin-homologous domain also undergoes autophosphorylation, but the kinase activity is not stimulated by Ca2+. Phosphoamino acid analysis showed that NtCDPK1 phosphorylates serine and threonine residues. Finally, a 60 kDa protein which matches the expected size of NtCDPK1 was immunodetected in the membrane fraction by an antiserum reacting with NtCDPK1. Immunoprecipitation and in vitro phosphorylation using the antiserum also generated a 60 kDa phosphoprotein only in the membrane fraction. These results suggest that NtCDPK1 is associated with the membrane.