Molecular Cloning And Characterization Of A Calcium-Depdendent Protein Kinase Isoform ScCPK1 From Swainsona Canescens

Abstract

Plants are constantly exposed to pathogens and various environmental stresses, such as cold, salinity and drought. Plants normally respond rapidly to these biotic and abiotic stresses. Efficient perception of biotic and abiotic stresses and cell programmed signaling mechanisms for appropriate responses are important for growth and survival of plants. Calcium is an important second messenger in signaling pathways that respond to environmental stresses, pathogen attack as well as hormonal stimuli (For review, see DeFalco et al., 2010; Reddy and Reddy, 2004; Sanders et al., 2002). The transient increase of cytosolic free calcium concentration has been shown in a variety of external signals (Reddy, 2001), which in turn triggers many signal transduction pathways leading to a variety of cellular responses (Bush, 1995). Any calcium mediated signal transduction process involves generation of signal-specific calcium signature in the cytosol (Scrase-Field and Knight, 2003). These changes in cytosolic calcium level or ‘calcium signatures’ are sensed by the specific group of proteins called the ‘calcium sensors’. Different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. In plants the protein kinases are a large and differentiated group of calcium sensors. After analyzing 1264 protein kinase sequences, a superfamily of protein kinase called CDPK/SnRK family of protein kinase were defined (Hrabak et al., 2003). CDPK/SnRK family of protein kinases encompasses five subfamilies viz., calcium-dependant protein kinases, (CPKs), calcium/calmodulin dependant protein kinases (CCaMKs), calmodulin-dependant protein kinases (CaMKs), CPKrelated kinases (CRKs), and SnF1 related kinase 3 (SnRK3) and are regulated by calcium directly or indirectly. Among these, in plants, calcium-dependant protein kinases (CPKs) are predominant calcium sensors, which are shown to be involved in myriads of physiological responses. They are Ser/Thr family of protein kinases typically made up of five domains with an Nterminal variable domain followed by catalytic protein kinase domain, an autoinhibitory/ junction domain, a regulatory calmodulin-like domain (CaMLD) and a Cterminal domain of variable length. The CPKs are unique due to the presence of CaMLD which couples the calcium sensor directly to its responder (kinase domain). Although CPKs are highly conserved, there are several features that distinguish different members of the plant CDPK family. In an attempt to investigate the role of a CPK isoform, in the present work we bring out the results and inferences on isolation and characterization of a novel cDNA encoding a calcium-dependant protein kinase isoform ScCPK1 from Swainsonacanescens, a pharmaceutically important Australian herb known to produce an anticancer drug, swainsonine. Initially, we have cloned an 800 bp partial CPK cDNA from S. canescens by reverse transcription polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers designed based on conserved regions of the other known CPKs. A 2.1 kb full length CPK was obtained using 5` and 3` RACE which was designated as ScCPK1. An open reading frame (ORF) of 1659 bp was detected that encodes a protein of 552 amino acids with a calculated molecular mass of 61.8 kDa. Comparison of the deduced amino acid sequence of ScCPK1 with sequences of other CPKs revealed the highest identity (>90%) to Glycine max and Vigna radiate CPKs. As described for other CPKs, ScCPK1 has a long variable domain (88 aa), an auto-inhibitory domain (31 aa) and a C-terminal calmodulin domain (145 aa) containing four EF-hand calcium binding motifs, which is found in many CPKs. Phylogenetic tree analysis showed that ScCPK1 was closely related to StCPK4 , CmCPK1 and CmCPK2. The entire coding region of ScCPK1 was cloned into pRSETA expression vector and expressed as fusion protein in E.coli. The recombinant ScCPK1 protein was purified to homogeneity by NiNTA affinity chromatography. The recombinant purified ScCPK1 was catalytically active in a calcium-dependent manner. The recombinant ScCPK1 phosphorylated itself and histone IIIS as substrate only in the presence of Ca2+. Phosphoaminoacid analyses showed that ScCPK1 phosphorylates serine and threonine residues of histone IIIS and its autophosphorylation also occurs on serine and threonine residues. ScCPK1 has a pH and temperature optima of 7.5 and 37 °C, respectively. It showed high affinity to histone III-S with a Km of 4.8 µM and had a Vmax of 4.700 pmoles of γ32P incorporation/min/mg at saturating substrate concentrations. The ScCPK1 is ~100fold active and showed 10fold higher affinities to histone III-S than CaCPK1 and CaCPK2, CPKs which were characterized from Cicer arietinum previously in our laboratory (Prakash and Jayabaskaran, 2006). From literature it is known that many CPKs are activated or inhibited by metal ions. (PutnamEvans etal., 1990; Anil and Rao, 2001). The influence of Na+ and Mg2+on the in vitro substrate phosphorylation activity of the recombinant ScCPK1 was tested in this work. Addition of NaCl strongly inhibited ScCPK1 activity. The inhibition of substrate phosphorylation activity by salt implies ionic interactions between the positively charged substrate and the enzyme’s active site. The optimum concentration of Mg2+ for ScCPK1 substrate phosphorylation activity was found to be 810 mM, similar to CaCPK1 and CaCPK2 (Prakash and Jayabaskaran, 2006). However, the activity was inhibited above 10 mM Mg2+suggesting the disruption of ionic interactions between the enzyme and the substrate. The kinase and autophosphorylation activities of the recombinant ScCPK1 were calmodulin independent and sensitive to CaM antagonists’ calmidazolium and W7 (N(6aminohexyl)5chloronaphthalene sulphonate). This indicates that the activation was supported by calmodulin-like domain, which is typical of CPK family. Farmer and Choi (1999), showed that DcCPK1 activity was inhibited by polyamines vizspermine and spermidine, and polylysine. We found that substrate phosphorylation activity of ScCPK1 was inhibited by polyLLysine with an IC50 of 8 M but not the polyamines, spermine and spermidine. An interesting aspect that makes CPKs attractive for research is their functional similarity to mammalian PKCs. There are no structural PKC analogues found in plant genomic data. Similar to PKCs, CPKs are regulated by intracellular Ca2+ signals. There is also experimental evidence that some of the CPKs are additionally activated by phospholipids (Farmer and Choi, 1999; Szczegielniak etal., 2000). We investigated the effects of lipid molecules on the activity of ScCPK1. Phosphorylation of histone IIIS by ScCPK1 was stimulated by phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol in the presence of Ca2+, where as lysophosphatidylcholine, phosphatidylcholine and phosphatidic acid did not increase the enzyme activity. Our data that shows interaction of ScCPK1 with phospholipids supports the idea that this protein kinase could be associated with the membrane. The work from Farmer and Choi (1999), with DcCPK1 suggested that some of the PKClike activities observed in plants may be attributed to CPKs. They also demonstrated that DcCPK1 phosphorylated PKC pseudosubstrate peptide and also was sensitive to staurosporine inhibition. However, the protein kinase inhibitor, staurosporine inhibited the substrate phosphorylation activity of ScCPK1 completely with an IC50 value of 700 nM invitro. But PKC inhibitor PMA was less effective, inhibiting the substrate phosphorylation activity of ScCPK1 to a maximum of 50%, but at a very high concentration (200 nM). Our data suggests that ScCPK1 may not have any features attributable to PKC. We investigated subcellular localization of the ScCPK1. To gain a better understanding of the subcellular localization of the ScCPK1, we generated GFP fusion protein of ScCPK1 and transiently expressed it in Agrobacterium-mediated transformed tobacco BY2 cells. Analysis of the GFP expression patterns in transformed tobacco BY2 cells revealed ScCPK1 localization in the plasma membrane of the transformed tobacco BY2 cells despite lacking consensus myristoylation and palmitoylation motifs (as per in silico analyses). Taking together, our data have demonstrated that ScCPK1 is a Ser/Thr protein kinase and its sub-cellular localization studies revealed that it is localized to plasma membrane. We propose that ScCPK1 is a key component of one or more signaling pathways and plays vital roles in plant development, responses to environmental stimuli and/ or in secondary metabolite biosynthetic gene expression. The involvement of the ScCPK1 as a component of signaling pathways warrants further studies.