ARTICLE

Cell Research, 15(8):604-612, August 2005

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

Received, May 10, 2005 Revised, July 26, 2005 Accepted, Aug 15, 2005

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 V_max and K_m 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 V_max of 2008 nmol/min/mg and a lower K_m of 30 µM. The K_0.5 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.

Keywords: calcium-dependent protein kinase, phosphorylation, GFP, subcellular localization.