Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco.
By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 (accession number AY971376), which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526 nmol/min/mg and 210 microg/ml respectively with calf thymus histone (fraction III, abbreviated to histone IIIs) as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30 microM. The K0.5 of calcium activation was 0.04 microM or 0.06 microM for histone IIIs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.
Wang
Yun
Y
Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Zhang
Mei
M
Ke
Ke
K
Lu
Ying Tang
YT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases