Research

CDPK IN PLANTS

Supported by the NSF MCB Program

Calcium-Dependent Protein Kinases (CDPKs) in Plants:

The first calcium-dependent protein kinase (CDPK) activities were reported in pea shoot membranes more than 20 years ago by Hetherington and Trewavas (1982). Harmon and colleagues provided the first biochemical evidence that soybean CDPKs are calmodulin independent (Harmon et al., 1987). The first CDPK cDNA clones were isolated by Harper and colleagues from Arabidopsis (Harper et al., 1991) and Suen and Choi from carrot (1991). CDPKs have now been identified throughout the plant kingdom from green algae to angiosperms (Hrabak, 2000; Harmon et al., 2001). Other than plants, CDPKs are found only in some protozoans, and are notably absent from the sequenced eukaryotic genomes of yeast, worms, flies, mice and humans (Harmon et al., 2000; Cheng et al., 2002; Hrabak et al., 2003). However, the PK domains of CDPKs are highly homologous to the mammalian multifunctional calmodulin-dependent PKs (CaMKII), suggesting a common evolutionary origin. Analysis of the Arabidopsis genome sequence indicates the presence of 34 CDPK genes (The Arabidopsis Genome Initiative, 2000; Harmon et al., 2000; Cheng et al., 2002; Hrabak et al., 2003). Information available from other genomic sequencing and extensive expressed sequence tag (EST) projects also indicates the presence of multigene families of CDPKs in other plants, including soybean, tomato, rice, and maize (Harmon et al., 2000). Future challenges will be to elucidate their specific or redundant physiological functions in plants.

 

The Roles of Arabiodopsis CDPKs in Early Defense Signal Transduction:

Plant cells appear to possess many sensors to detect non-self MAMPs/PAMPs (microbe-/pathogen-associated molecular patterns) and launch convergent as well as specific defense responses. An Arabidopsis leaf cell system based on the induction of early defense gene transcription by diverse MAMPs/PAMPs, including flagellin, LPS (lipopolysaccharide), harpin, NPP1 (necrosis-inducing Phytophthora protein 1) and chitin, has be established to study innate immune signaling in plants. Previous studies have identified a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) and WRKY22/29 transcription factors that function downstream of the flagellin receptor FLS2, a LRR receptor kinase. Using the same system, we have also discovered "MAPK-independent" signaling pathways controlled by a group of CDPKs in innate immune responses. In addition, some CDPKs also play important roles in specific gene-for-gene (AvrRpt2-RPS2 & AvrB-RPM1/AvrRmp1-RPM1) mediated early signaling. We have hypothesized that CDPKs with different expression patterns, subcellular localizations and substrate specificity can perceive overlapping or/and distinct calcium signals triggered by early defense responses, and alter transcription in the nucleus and other cellular activities. The cell-based system and reverse genetics will be used to determine the precise roles of multiple CDPKs in convergent and specific signaling events initiated by diverse pathogen signals.

 

References:

1. Cheng SH, Willmann MR, Chen HC, and Sheen J (2002) Calcium Signaling through Protein Kinases. The Arabidopsis Calcium-Dependent Protein Kinase Gene Family. Plant Physiol. Vol. 129 469-485
2. Harmon AC, Putnam-Evans C, Cormier MJ (1987) A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol 83: 830–837
3. Harmon AC, Gribskov M, Gubrium E, Harper JF (2001) The CDPK superfamily of protein kinases. New Phytol 151: 175–183
4. Harmon AC, Gribskov M, Harper JF (2000) CDPKs: a kinase for every Ca2+ signal? Trends Plant Sci 5: 154–159
5. Harper JF, Sussman MR, Schaller GE, Putnam-Evans C, Charbonneau H, Harmon AC (1991) A calciumdependent protein kinase with a regulatory domain similar to calmodulin. Science 252: 951–954
6. Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu JK, Harmon AC. (2003) The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680
7. Hrabak EM (2000) Calcium-dependent protein kinases and their relatives. In M Kreis, JC Walker, eds, Advances in Botanical Sciences, Plant Protein Kinases, Vol 32. Academic Press, New York, pp 185–223
8. Hetherington AM, Trewavas A (1982) Calcium-dependent protein kinase in pea shoot membranes. FEBS Lett 145: 67–71
9. Suen KL, Choi JH (1991) Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin. Plant Mol Biol 17: 581–590

 
back to top
 

Summary of Arabidopsis CDPKs Characteristics

 

Domain Structure

Four distinct domains typify CDPK family members: an N-terminal variable domain, a protein kinase domain, an autoinhibitory domain, and a calmodulin- like domain (Fig. 1). Based on phylogenetic analysis, it is thought that the CDPK gene family arose through the fusion of a CaMK and a calmodulin (Harper et al., 1991; Suen and Choi, 1991; Harmon et al., 2000; Zhang and Choi, 2001). This unique molecular structure allows the direct activation of CDPKs by Ca2+. Unlike the analogous mammalian protein, the multisubunit CaMKII, CDPKs function as monomers (Roberts and Harmon, 1992).

 

Figure 1. Structural comparisons of mammalian CaMKII and plant CDPKs. The kinase domain of CDPKs is up to 44% identical (65% similar) to that of mouse (Mus musculus) CaMKII alpha (accession no. S04365) and 43% identical (65% similar) to that of mouse CaMKII beta (accession no. NP_031621). N, Amino-terminal variable domain; K, kinase domain; A, autoinhibitory domain; CaM, calmodulin. The four bars within the CaM-like domain represent the EF hand Ca2-binding sites.

 

Sequence Homology

 

All 34 Arabidopsis CDPKs are highly homologous to each other. Pair-wise analyses with the full protein sequences indicate that the overall identities and similarities are 39% to 95% and 56% to 96%, respectively. High identities are found between AtCPK 4 and 11 (95%), AtCPK 17 and 34 (93%), AtCPK 7 and 8 (90%), AtCPK 10 and 30 (86%), AtCPK 9 and 33 (85%), AtCPK 1 and 2 (81%), and AtCPK 21 and 23 (81%), and among AtCPK 5, 6, and 26 (85%–88%). Because both AtCPK 10 and 30 specifically activate a stress pathway (Sheen, 1996), such high homologies may indicate similar functions. AtCPK16, 18, and 28 are the most divergent CDPKs, as indicated by their relatively low average pair-wise identity/similarity values (45% and 64%, respectively). Based upon sequence homology, the CDPKs of Arabidopsis cluster into four subgroups (I–IV). Subgroup IV is the least complex, with three members, and subgroup II is the most complex, with 13 members. This pattern of grouping was also found when the tree was constructed based on the sequences of the kinase domain only (data not shown; Harmon et al., 2001). Subgroups I through III are closer in sequence identity to each other than to subgroup IV. It is not known whether such a pattern of clustering reflects any functional differences between the subgroups.

 
Protein Sequence Alignment of 34 Arabidopsis CDPKs
 
Protein Sequence Similarity of 34 Arabidopsis CDPKs: Full Length, Kinase Domain , Autoinhibitory Domain, Calmodulin Domain
 
Figure 2. Relatedness of Arabidopsis CDPKs. The complete protein sequences of the AtCPKs were aligned and analyzed by the Treeview 1.6.5 program (http:/taxonomy.zoology.gla.ac.uk/rod/rod.html). The unrooted distance tree reveals the presence of four distinct, branched subgroups (I–IV). The branch lengths are proportional to divergence, with the scale of “0.1” representing 10% change.
 

Chromosomal Distribution

 

The 34 Arabidopsis CDPKs are distributed among all five chromosomes (Fig. 3). Chromosome IV has the most CDPKs (11), whereas chromosome III has the least (4). The only region that contains no CDPKs is the short arm of chromosome II. Interestingly, one gene cluster on the short arm of chromosome IV contains five genes (AtCPK 21, 22, 23, 27, and 31), all within subgroup IV. They are organized in tandem in the same transcriptional orientation (Fig. 3), and their amino acid sequences are very homologous (61%– 82% identity and 74%–89% similarity). Furthermore, sequence homology also exists in the N-terminal variable domain in this gene cluster (21%–78% identity and 22%–85% similarity). These results suggest that they arose relatively recently by gene duplication and that they may have similar or overlapping functions.

 

Figure 3. Genomic distribution of CDPKs on the Arabidopsis chromosomes. Ovals on the chromosomes indicate the location of the centromeres. The arrows next to the gene names show the direction of transcription. The numbers in parentheses designate the position of the first exon of each CPK gene in megabases (Mb). The chromosome numbers are indicated by Roman numerals.

 
back to top

CDPK Substrates

Although CDPKs have been shown to phosphorylate a large number of protein substrates in vitro (Table II), limited information is available regarding substrate specificity and phosphorylation sites in vivo. Although CDPKs are highly homologous, an examination of in vitro substrate phosphorylation by spinach and soybean CDPKs suggests that CDPKs will exhibit substrate specificity differences in vivo (Bachmann et al., 1996; Lee et al., 1998). The use of synthetic peptides has facilitated delineation of the potential phosphorylation motifs recognized by some CDPKs.

 
Known potential CDPK substrates
 
back to top

CDPK T-DNA Insertional Mutants

Gene disruption and silencing techniques, such as insertional mutagenesis, RNA interference, and virusinduced gene silencing, can be used to study altered phenotypes (Krysan et al., 1996; Waterhouse et al., 1998; Romeis et al., 2001). Sequencing of publicly and privately generated insertion mutants of Arabidopsis has already identified a number of putative CDPK mutants, with others rapidly becoming available. Click here for the list of CDPK T-DNA lines currently available from SALK and Syngenta.

 
back to top
 

Reviews & Research Papers

Boudsocq, M., Willman, M., McCormack, M., Lee, H., Shan, L., He., P., Bush, J., Cheng, S.H., and Sheen, J. 2009. Differential innate immune signalling via Ca2+ sensor protein kinases. Nature.

Boudsocq, M. and Sheen, J. 2009. Calcium Sensing and Signaling. In: Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic Foundation (A.Pareek, S.K. Sopory, H.J. Bohnert, Govindjee, eds). Springer, Dordrecht, The Netherlands: Springer PDF

Fujii, H., Chinnusamy, V., Rodrigues, A., Rubio, S., Antoni, R., Park, S.-Y., Cutler, S.R., Sheen, J., Rodriguez, P.L. and Zhu, J.-K. 2009. In vitro reconstitution of an abscisic acid signaling pathway. Nature. 462: 660-664. PDF SUPP

Yoo, S.-D., Cho, Y.-H., and Sheen, J. 2007. Arabidopsis Mesophyll Protoplasts: A Versatile Cell System for Transient Gene Expression Analysis. Nature Protocols 2:1565-1572 PDF

Shan, L., He, P., and Sheen, J. 2007. Endless Hide-and-Seek: Dynamic Co-evolution in Plant-Bacterium Warfare. J.Integr. Plant Biol. 49(1):105-111 PDF

He, P., Shan, L., and Sheen, J. 2007. Elicitation and Suppression of Microbe-Associated Molecular Pattern-Triggered Immunity in Plant-Microbe Interactions. Cellular Microbiology 9:1385-1396 PDF

He, P., Shan, L., and Sheen, J. 2006. The use of protoplasts to study innate immune responses. Elicitation and Suppression of Microbe-Associated Molecular Pattern-Triggered Immunity in Plant-Microbe Interactions. Methods Mol. Biol. 354:1-10 PDF

Cheng, S.-H., Willmann, M. R., Chen, H.-C. and Sheen. J., 2002, Calcium Signaling through Protein Kinases: The Arabidopsis Calcium-Dependent Protein Kinase Gene Family, Plant Physiol. 129: 469-485 PDF

Cheng, S.-H., Sheen, J., Gerrish, C., and Bolwell, G.P. 2001. Molecular identification of phenylalanine ammonia-lyaseasa substrate of a specific constitutively active Arabidopsis CDPK expressed in maize protoplasts. FEBS Lett. 503(2-3):185-8. PDF

Cheng, W.-H., Endo, A., Zhou, L., Penney, J., Chen, H.-C., Arroyo, A., Leon, P., Nambara, E., Asami, T., Seo, M., Koshiba, T., and Sheen, J. 2002. A Unique Short-Chain Dehydrogenase/Reductase in Arabidopsis Glucose Signaling and Abscisic Acid Biosynthesis and Functions. Plant Cell 14 2723-2743 PDF

Shen, Q, Gomez-Cadenas, A., Zhang, P., Walker-Simmons, M. K., Sheen. J., and Ho, T.H., Dissection of abscisic acid signal transduction pathways in barley aleurone layers. Plant Mol.Biol.. 47: 437-448 PDF

Sheen, J. 2001. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 2001 Dec;127:1466-1475. PDF

Sheen, J. 1999. C4 gene expression. Ann Rev Plant Physiol Plant Mol Biol. 50: 187-217. PDF

Sheen, J. 1998. Mutational analysis of two protein phosphatases involved in ABA signal transduction in higher plants. Proc. Natl. Acad. Sci. USA. 95:975-980. PDF

Saijo Y, Hatta, S., Sheen, J., and Izui, K. 1997. cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases. Biochim Biophys Acta. 1350 (2):109-14.

Sheen, J. 1996. Specific Ca2+-dependent protein kinase in stress signal transduction. Science 274: 1900-1902. PDF

Chiu, W.-L. , Niwa, Y, Zeng, W, Hirano, T., Kobayashi, H, Sheen, J. 1996. Engineered GFP as a vital reporter in plants. Current Biol. 6: 325-330. PDF

Sheen, J., Schäffner, A.R., Leon, P., To, K.-Y., and Jang, J.-C. 1996. C4 genontrolled by diverse mechanisms and signaling pathways. 1996 Robertson Symposium, In " C4 Photosynthesis", Australia, p. 5-6

Shen, Q., Zhang, P., T.-H.D. Ho, and Sheen, J. 1996. Involvement of PP2C in the signaling transduction of ABA action in barley aleurone cells. The 15th Annual Missouri Symposium. In Current Topics in Plant Biochemistry, Physiology and Molecular Biology. pp. 42-43

Sheen, J. 1994. Blue light signaling in maize leaf cells. In "Responses of the photosynthetic apparatus to environmental light conditions". The 34th NIBB Conference. Japan. pp124-125

Sheen, J. 1993. Protein phosphatase activity is required for light-inducible gene expression in maize. EMBO J. 12: 3497-3505.

Sheen, J. 1991. Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell. 3: 225-245.

Schäffner, A.R., Sheen, J. 1991. Maize rbcS promoter activity depends on sequence elements not found in dicot rbcS promoters. Plant Cell 3: 997-1012.