Biochemical And Expression Analyses Of New Isoforms Of Calcium-dependent Protein Kinase From Tobacco

Two full-length cDNA clones were isolated from tobacco (Nicotiana tabacum) with maize MCK1 as probe. Sequence analyses showed that these two cDNAs encoded high homologous calcium-dependent protein kinases, named NtCPK4 and NtCPK5. This project focused on the sequences analyses, biochemical characterization, analyses of expression pattern and subcellular localization about these two protein kinases. First, sequence analyse showed that NtCPK4 and NtCPK5 were both the evolutionary link between CPKs and CRKs. The biochemical assay indicated that their kinase activities were dependent upon the free Ca²⁺, and the phosphoamino acid of autophosphorylation NtCPK4 was threonine identified by capillary electrophoresis. Then we analysed one protein kinase gene, NtCPK4's expression pattern through RNA in situ hybridization and Northern blot. The probe in these two assays was made with the 3' untranslational region of NtCPK4 cDNA to avoid the non-specific hybridization signal. With GFP as a tag, the transient expression assay in onion epidermal cells was carried out to identify the subcellular localization of these two protein kinase. The transformations were made with genegun. Our results showed that NtCPK4 and NtCPK5 have absolutely different distribution in onion epidermal cells and NtCPK5 anchored at membrane mostly. Further experiment with site-mutated NtCPK5 indicated that the two N-terminal amino acid residues played vital roles in the subcellular localization of NtCPKs.1. The full-length cDNA of 2360 bp contains an open reading frame for NtCPK4 consisting of 572 amino acid residues, with 198 bp untranslantional region, 1719 bp open reading frame, and 443 bp 3' untranslantional region, while the cDNA of NtCPK5 is 2368 bp coding 567 amino acid residues, and its 5' untranslantional region, open reading frame and 3' untranslantional region were 247 bp, 1704 bp and 417 bp respectively. Both of the amino acid sequences of NtCPK4 and NtCPK5 have the conservative domains of calcium-dependent protein kinase, including kinase domain, autoinhibitory region (pseudo substrate), andCaM-like domain (Ca2+-binding domain). Sequence alignment indicated that NtCPK4 and NtCPK5 shared high similarities with other CPKs and some CPK-related protein kinases (CRKs), so we proposed that both of them are evolutional links between CPKs and CRKs.2. To identify whether the protein kinases' activities are dependent on free Ca+ biochemically, we constructed the recombinant viruses with which transfected Sf9 cells and purified the recombinant proteins from the cells. Then the purified full-length or truncated proteins of NtCPK4 and NtCPK5 were used in kinase assay to analyse the kinetic properties. The results showed that these two NtCPKs' activities of autophosphorylation or substrate phosphorylation were dependent on Ca2+ with histone Ills or syntide-2 as substrate. The kinase activities with histone Ills or syntide-2 as substrate in different concentration were measured and fitted to a Lineweaver-Burk curve. When histone Ills as substrate, Km value of NtCPK4 was 176 ug ml'1 and Kmax of NtCPK4 was 588 nmol min"1 mg"1. While syntide-2 as substrate, NtCPK4 had a Km value of 58 uM and Vm!tx value of 2415 nmol min"1 mg'1. The Km and Fmax value of NtCPK5 were 210 jig ml*1 and Vmax was 526 nmol min*1 mg'1 with histone Ills as substrate, and they were 30 uM and 2008 nmol min"1 mg"1 accordingly with syntide-2 as substrate. In addition, the phosphorylation of NtCPK4 occurred on threonine residue as shown by capillary electrophoresis analyses. To investigate the optimum reaction conditions for two NtCPKs, range of Mg2+ concentration (0 - 25 mM) and pH (6 - 9.5) in kinase buffer were detected for NtCPK5 activities. The time-course assays were performed to detect the kinase reaction rate. Different substrates were used to detect the effect of Ca2+ concentration for NtCPKs' activity. When histone Ills as substrate, the activity of NtCPK4 was stimulated by the increasing concentration of Ca2+ and one-half maximal activation (Ko.s) was 0.29 uM, while syntide-2 as substrate, the Ca2+ concentration for one-half maximal activation was 0.25 uM. The corresponding values of NtCPKS were 0.04 uM and 0.06 uM. These results showed that NtCPK4 and NtCPK5 were typical calcium-dependent protein kinases biochemically.3. NtCPK4 as a low copy gene was expressed in all tested organs including root, leaf, stem and flower of tobacco while its expression was temporally and spatially modulated in both productive and vegetative tissues during tobacco growth and development. NtCPK4 expression was also increased in response to the treatment of gibberellin or NaCl. Our study suggested that NtCPK4 might play vital roles in plant development and responses to environmental stimuli. The localization of the NtCPK4's transcriptions accumulated in the tissues/cells that were rapidly growing, dividing and metabolizing. These sites included the developing embryo, the stigma, the root tip and lateral root primordia, the shoot apical meristems and lateral branch primordia, the vascular in leaf or anther, and sporogenous cells,suggesting that it might be related to cell differentiation and metabolism.4. Because of the high homology between the amino acid sequences, and the variety of the N-termini of NtCPK4 and NtCPK5, we presumed that they have different localizations in cells. A transient expression assay was performed in onion epidermal cells with genegun transformation. Results showed that the GFP as a control was distributed evenly in whole cell, while the NtCPK4/GFP was efficiently targeted to cytoplasm and nuclear and still little was distributed at membrane, but difference between these two kinds distribution of GFP and NtCPK4/GFP was not clear. While bombarding onion epidermal cells with NtCPK5/GFP vectors, we could find that wild-type NtCPK5/GFP was efficiently targeted to the cell periphery, which could be presumed anchoring on membrane. Because NtCPK5 had presumed N-terminal acylation sites while NtCPK4 did not, so we proposed that these sites played vital roles in these different subcellular localizations. This presume could be also confirmed by site-mutation to Ala at the second Gly, the forth Cys, or both of them in NtCPK5. The mutated NtCPK5/GFP fusion proteins were not targeted to the plasma membrane. These data demonstrate that both N-terminal myristoylation and palmitoylation are essential for plasma membrane targeting of NtCPK5 in onion cells. N-myristoylation was not enough for NtCPK5 membrane localization but might be important to further promote subsequent palmitoylation, a dynamic process that could regulate the reversible subcellular distribution of this kinase.