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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. |
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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. |
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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 |
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Summary
of Arabidopsis CDPKs Characteristics |
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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). |
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| 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. |
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Sequence
Homology |
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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.
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Protein
Sequence Alignment of 34 Arabidopsis CDPKs |
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Protein Sequence Similarity of 34 Arabidopsis
CDPKs: Full
Length, Kinase
Domain , Autoinhibitory
Domain, Calmodulin
Domain |
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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. |
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Chromosomal
Distribution |
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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. |
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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. |
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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. |
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Known potential CDPK substrates |
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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. |
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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.
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