Volume 15, No 8, Aug 2005
ISSN: 1001-0602
EISSN: 1748-7838 2018
impact factor 17.848*
(Clarivate Analytics, 2019)
Volume 15 Issue 8, August 2005: 604-612
ORIGINAL ARTICLES
Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco
Yun Wang¶, Mei Zhang¶, Ke KE, Ying Tang Lu*
Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China
Correspondence: Ying Tang LU(yingtlu@whu.edu.cn)
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 µg/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 µM. The
K0.5 of calcium activation was 0.04 µM or 0.06 µM 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.
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