article addendum
article addendum
Plant Signaling & Behavior 5:8, 991-994; August 2010; © 2010 Landes Bioscience
Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts
with MEKK1 in plants
Tianbao Yang,1,† Gul Shad Ali,2 Lihua Yang,1 Liqun Du,1 A.S.N. Reddy2 and B.W. Poovaiah1,*
1
Center for Integrated Biotechnology and Department of Horticulture; Washington State University; Pullman, WA USA; 2Department of Biology
and Program in Molecular Plant Biology; Colorado State University; Fort Collins, CO USA
†
Current address: Food Quality Laboratory; USDA-ARS; Beltsville, MD USA
R
Key words: calcium, calmodulin, cold
stress, MAPK, Arabidopsis, protein phosphorylation
Submitted: 04/27/10
Accepted: 04/28/10
Previously published online:
www.landesbioscience.com/journals/psb/
article/12225
*Correspondence to: B.W. Poovaiah;
Email: [email protected]
Addendum to: Yang T, Chaudhuri S, Yang L, Du
L, Poovaiah BW. A calcium/calmodulin-regulated
member of the receptor-like kinase family confers cold tolerance in plants. J Biol Chem 2010;
285:7119–26; PMID: 20026608; DOI: 10.1074/jbc.
M109.035659.
www.landesbioscience.com
ecently we reported that CRLK1,
a novel calcium/calmodulin-regulated receptor-like kinase plays an
important role in regulating plant cold
tolerance. Calcium/calmodulin binds
to CRLK1 and upregulates its activity.
Gene knockout and complementation
studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we
show that MEKK1, a member of MAP
kinase kinase kinase family, interacts
with CRLK1 both in vitro and in planta.
The cold triggered MAP kinase activation in wild-type plants was abolished
in crlk1 knockout mutants. Similarly,
the cold induced expression levels of
genes involved in MAP kinase signaling
are also altered in crlk1 mutants. These
results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in
plants.
Calcium, a universal second messenger
in eukaryotic cells, mediates changes in
external and internal signals leading to
the physiological responses.1-4 Calcium/
calmodulin (Ca 2+/CaM)-dependent protein kinases (CaMKs) are very important
players in calcium/calmodulin mediated
signaling in mammalian cells.5 In plants,
Ca 2+/CaM-dependent protein phosphorylation was observed more than 25
years ago.6 Several calmodulin-regulated
protein kinases have been identified and
characterized.7,8 For example, plants have
a unique chimeric Ca 2+/CaM-dependent
protein kinase (CCaMK), which exhibits
Ca 2+ -dependent autophosphorylation and
Ca 2+/CaM-dependent substrate phosphorylation.9 CCaMK is required for bacterial and fungal symbioses in plants.10-12
Recently, we characterized a novel plantspecific calcium/CaM-regulated receptorlike kinase, CRLK1.13 Ca 2+/CaM binds to
CRLK1 and stimulates its kinase activity.
Functional studies with CRLK1 indicate
that CRLK1 acts as a positive regulator
in plant response to chilling and freezing temperatures. To further define the
CRLK1-mediated signal pathway, we
isolated CRLK1 interacting proteins by
co-immunoprecipitation using an antiCRLK1 antibody. Since cold increases
the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C
for 1 hr before co-immunoprecipitation.
The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We
observed several bands of different sizes
only in the wild-type but not in the
crlk1 knockout mutant plants (Fig. 1A).
Furthermore, the intensity of these bands
increased upon cold treatment, suggesting that they are the putative partners
or associated proteins of the CRLK1
immunocomplex.
To determine the identities of these
proteins, mass spectrometric analysis
was performed with the total immunocomplex.14 In addition to CRLK1, there
were 12 other proteins which matched
the Arabidopsis database. Several of them
appeared in the pull-down complex from
WT, but not from crlk1 mutants. These
putative interacting proteins included
MEKK1, another unknown protein
Plant Signaling & Behavior991
Figure 1. CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or
without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by
MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CRLK associates with MEKK1 in
vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.
kinase, a type 2C phosphatase and CaM
(Fig. 1B). MEKK1 is one of the 60
putative MAPKKKs in the Arabidopsis
genome, and sits on the top of mitogenactivated protein kinase (MAPK) cascade. The MAPK signaling consists of
a cascade of three consecutively acting
protein kinases, a MAP kinase kinase
kinase (MAPKKK), a MAP kinase kinase
(MAPKK) and a MAP kinase (MAPK).
Plants possess multiple MAPKKKs,
MAPKKs and MAPKs, which respond
to different upstream signals and activate
distinct downstream pathways.15-17 The
specific MAPK module responding to
lower temperature has been determined
in Arabidopsis.18,19 MEKK1, a member
of MAPKKKs, specifically interacts and
phosphorylates MKK2 and regulates
COR genes expression in response to cold
stress.19 MEKK1 has been shown to play a
role in mediating reactive oxygen species
992
homeostasis.20,21 Therefore we selected
MEKK1 from the putative CRLK1 partners for further studies.
CRLK1 Interacts With MEKK1
in vitro and in planta
To confirm the direct interaction between
CRLK1 and MEKK1, a well-characterized component in cold signaling, we performed GST pull-down assay (Fig. 1C).
The recombinant CRLK1 M29-440 was
precipitated by GST:MEKK1, but not
by GST alone. However, the intensity of
the band was very low, suggesting weak
interaction between them. Since CRLK1
is a calcium/CaM-regulated kinase, we
investigated the effects of calcium and/or
CaM on the interaction between CRLK1
and MEKK1. In the presence of calcium
and CaM in the reaction mixture, the
interaction between CRLK1 and MEKK1
Plant Signaling & Behavior
was dramatically increased as reflected by
the intensity of the band (Fig. 1C). These
results indicate that the binding of calcium/CaM to CRLK1 increases its affinity to MEKK1.
To address if CRLK1 and MEKK1
associate in vivo and to determine subcellular location of this association,
we used Bimolecular Fluorescence
Complementation (BiFC) in Arabidopsis
protoplasts.22 BiFC vectors carrying CRLK
and MEKK1 were co-transfected into protoplasts and observed for the reconstitution of YFP fluorescence. Confocal images
showed that CRLK1 associated with
MEKK1 both on cell membranes and in
intracellular vesicle-like structures (Fig.
1D). Control co-transfections with nEYFP
or cEYFP vectors did not display any fluorescence suggesting that the interaction
between CRLK1 and MEKK1 is specific
(Fig. 1D).
Volume 5 Issue 8
Loss of CRLK1 Altered
MAP Kinase Activity and
Expression Levels of Genes
Involved MAPK Pathway
To further study the relationship between
CRLK1 and MAPK signaling, we compared the MAPK activity between WT
and crlk1 plants in response to cold treatment using in gel phosphorylation assay
(Fig. 2A). In WT plants, cold stimulated
MAP kinase activity. However, this stimulation was diminished in crlk1 mutants.
These results suggest that CRLK1 plays a
role in regulating the MAPK cascade during cold signaling.
It has been shown that cold treatment
increases the expression of MEKK1.23
To investigate whether CRLK1 affects
MEKK1 expression, we compared the
RNA level of MEKK1 between WT and
crlk1 mutants using semi-quantitative
RT-PCR. MEKK1 levels in both WT and
crlk1 plants were similar after cold treatment, suggesting that CRLK1 does not
regulate MEKK1 at the transcriptional
level (data not shown). We further studied the expression of the marker genes
such as RAV1, RAV2 and STZ affected
by MAPK cascade.19 Their expression
levels were lower in crlk1 plants as compared to WT after cold treatment (Fig.
2B). These results are consistent with our
earlier observation that CBF and COR
genes expression were reduced or delayed
in crlk1 knockout plants as compared to
wild-type plants during cold treatment.13
It is documented that cold responsive CBF
and COR genes expression are regulated
by MAPK pathway.19
Accumulating evidence indicates that
protein phosphorylation is involved in the
pathway connecting the cold-triggered
calcium changes and cold acclimation.24,25
However, the protein kinase(s) responsible for inducing cold-regulated genes and
activating freezing tolerance is elusive.
There are interesting candidates such as
an alfalfa MPK, p44mmk4, that is activated
within 10 min after being exposed to low
temperature.26 Expressing a heterologous
tobacco MAPKKK (Nicotiana PK1)
enhances freezing tolerance in transgenic
maize plants that are normally frost sensitive.27 In Arabidopsis, MEKK1, MKK2,
MPK4 and MPK6 have been shown to be
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Figure 2. CRLK1 regulates the MAPK kinase activity and expression levels of genes affected by
MAPK cascade during cold treatment. (A) In gel phosphorylation showed that cold stimulated
MAP kinase activity is diminished in crlk1 plants. The in-gel assay of endogenous MAPK was carried out as described.28 Briefly, 50 µg of plant protein was fractioned in a 12.5% SDS-PAGE with
0.25 mg/ml mylein basic protein (MBP). The gel was incubated with protein phosphorylation
buffer with [γ32P-ATP], and exposed to X-ray film after washing. Forty micrograms of total proteins
were used in the assay. The Coomassie stained RUBISCO band was used to show equal loading.
(B) RT-PCR analysis of RAV1, RAV2 and STZ gene expression. Actin8 was used as an internal control.
(C) A hypothetical model showing cold stress signal transduction pathway linking calcium/calmodulin, CRLK1 and MAPK cascade.
involved in cold signal transduction.18,19,23
Our results suggest that calcium/CaM/
CRLK1 interacts with MEKK1 and regulates the MAPK cascade during cold stress.
Figure 2C is a working model showing
the components of CRLK1-mediated cold
stress signal transduction pathway linking calcium/calmodulin and MAPK cascade. Further studies on the hierarchy of
calcium/CaM/CRLK1 and MAPK will
shed light on our understanding of the
mechanisms of cold signal transduction
pathway(s) in plants.
Acknowledgements
We thank Dr. Shubho Chaudhuri for his
help and suggestions and Ms. Y. Chen for
her help in the laboratory. This project
was supported by the National Research
Initiative Competitive Grant no. 200835100-04566 from the USDA National
Institute of Food and Agriculture; grant
no. ISO-0642146 from the National
Science Foundation; the Washington State
Plant Signaling & Behavior
University Agricultural Research Center;
Colorado State University Academic
Enrichment Program grant 180470; and
by a National Science Foundation grant
(DBI 0743097).
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