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

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which we designated NtCDPK1. The deduced amino acid s equence of NtCDPK1 suggests that this protein contains the kinase domain at the amino terminus and the autoregulatory and calmodulin-like domains at t he carboxy terminus. NtCDPK1 is highly homologous to DcCPK1, a CDPK of carr ot, showing 76.5% amino acid sequence identity. NtCDPK1 transcripts are pre sent 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, chitosa n, and NaCl. The recombinant full-length NtCDPK1 protein is catalytically a ctive and highly stimulated by Ca2+. A truncated recombinant NtCDPK1 which lacks the C-terminal calmodulin-homologous domain also undergoes autophosph orylation, but the kinase activity is not stimulated by Ca2+. Phosphoamino acid analysis showed that NtCDPK1 phosphorylates serine and threonine resid ues. Finally, a 60 kDa protein which matches the expected size of NtCDPK1 w as immunodetected in the membrane fraction by an antiserum reacting with Nt CDPK1. Immunoprecipitation and in vitro phosphorylation using the antiserum also generated a 60 kDa phosphoprotein only in the membrane fraction. Thes e results suggest that NtCDPK1 is associated with the membrane.