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Ca2+ dependent phosphorylation modulates the activity of the ABA responsive transcription factor ABF2
Kai H. Edel, Katia Becker, Philipp Köster and Jörg Kudla Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 4, 48149 Münster, Germany.
Introduction
Results
Conclusion and Model
References
CIPK3.3-GFP ABF2-mCherry overlay
CFP BiFC
CB
L1
-
+
Intracellular Ca2+ elevation is one of the key events in plant response to various stresses 1. Besides fast reactions like the regulation of ion homeostasis or ROS, Ca2+ dynamics are associated with the control of gene transcription. Several motifs (including ABA responsive elements - ABRE) have been shown to be Ca2+ regulated 2,3. However, the signaling cascades leading to differential gene expres-sion upon Ca2+ elevation largely remain unknown. We combined in vitro protein biochemical methods and in vivo signaling pathway reconstitution methods to investigate the role of CBL/CIPKs in regulating ABRE mediated gene transcription by the transcription factor ABRE binding factor 2 (ABF2). Here we suggest the integration of phosphorylation and dephosphorylation events in the regulation of gene transcription as a convergence point of ABA and Ca2+ signaling
Kai Edel: E-mail: kai.edel@uni-muenster.de Phone +49 251 83 23325
IBBP - AG Kudla - Schloßplatz 4 48149 Münster - Germany
EF1 EF2 EF3 EF4
Kinase domain NAF
Regulatory domain Activation
loop
CBL
CIPK
1. CIPK3 and ABF2 localize in the nucleus
2. CIPK3 and ABF2 interact CBL1 dependently
9. CIPK3 phosphorylation sites are con- served in ABF-type transcription factors
Contacts
10. ABI1 dephosphorylates ABF2 in vitro
5. CIPK3 activates ABF2 Ca2+ dependently in protoplasts
Calcineurin B like proteins (CBLs) are a Bikonta specific group of Ca2+ sensor proteins harboring four po-tentially Ca2+ binding EF-hands. Differential fatty acid modification at the N-terminus is responsible for the subcellular localization of CBLs 4. They interact and thereby transmit Ca2+ signals to their interacting pro-tein kinases (CIPKs). CIPKs consist of a N-terminal kinases domain including the activation loop and a C-terminal regulatory domain. The NAF domain as part of the regulatory domain is a CIPK specific feature that is responsible for CBL interaction. Together CBL/CIPKs form sensor responder modules that regulate diverse Ca2+ responses 1.
normalized BiFC-Interaction quantification -mean FluorescenceCIPK3.3-YN + ABF2-YC
CBL1
CBL1EF
2/4 PM
0
5
10
15*
**
rel
mean
Flu
.
Outlook
0 2 4 6 8
2+Ionophore + ABA+ Ca
2+Ionophore + Ca
2+ABA + Ca
ABA
2+Ca
controlABF2 + CIPK3.3 + CBL9
LUC/GUS
0 20 40 60
+CBL1EF2/42+Ionophore+Ca
+CBL1EF42+Ionophore+Ca
+CBL12+Ionophore+Ca
CBL1
2+Ionophore + Ca
LUC/GUS
ABF2 + RCAR1 + CIPK3.3
6. ABF2 activation by CIPK3/CBL1 requires CBL Ca2+ binding
7. CBL1 plasma membrane targeting is required for ABF2 activation by CIPK3 but not for interaction
Fluorescence images of transiently transformed N. ben-thamiana epidermal cells.
BiFC interaction study and fluorescence quantification of transiently transformed N. benthamiana epidermal cells.
Schematic overview of the four phosphorylation sites found by mass spectrometric analyses and their con-servation among related transcription factors. The high conservation suggests further targets for CIPKs.
Both, Ca2+ elevation and ABA are required for full activation ofABF2 by CIPK3/CBL modules.
Co-expression of CBL1 variants with mutated EF-hands 2 and 4. Mutations prevent Ca2+ binding. Functional EF-hands are required for ABF2 activation.
Co-expression of CBL1 variant with mutated lipid modification sig-nal. Mutation prevents myristoylation at the N-terminus and there-by plasma membrane targeting. Lipid modification is required for ABF2 activation.
Fluorescence quantification (see 2. for details) of CIPK3 and ABF2 BiFC interaction in dependency of CBL1, CBL1EF2/4 or PM-OFP.
Fluorescence quantification (see 2. for details) of CIPK3 and ABF2 BiFC interaction in dependency of CBL1G2A or PM-OFP. Lipid modifica-tion is not required for interaction.
Autoradiograph of in vitro phosphorylation/dephos-phorylation assay. ABI1 is able to dephosphorylate ABF2. Addition of the ABA receptor PYL8 and ABA can inhibit the dephosphorylation.
ABF2 C1 C3 C4C2 bZIPP P P P
S26 S86/94 T135 S413
ABI5ATBZIP67AREB3ATBZIP12ABF2ABF3ABF4ABF1
Minimal 35S LuciferaseATG4x ABRE
(ACACGTGTA)-70 Bp
3. Reconstitution of a Ca2+ dependent signaling pathway in yeast
Schematic representation of the construct that was stably integrated into the yeast LYS gene 2.
ABF2 is activated by the co expression of CIPK3 and CBL1.
Protein extractionIncubation
Ca2+
IonophoreABA Incubation
Transformation
LUCGUS
0 20 40 60
+CBL1EF2/42+Ionophore+Ca
+CBL1EF42+Ionophore+Ca
+CBL12+Ionophore+Ca
CBL1
2+Ionophore + Ca
LUC/GUS
2 - 4 h 3 - 6 h
pRD29B LUC
35S GUS
35S ABF2
35S CIPK3
35S CBL1
Reporter
Control
Effector
Modulator
Modulator
Combine
4. Reconstitution of a Ca2+ dependent signaling pathway in vivo
• Study the effects of ABF2 p-site modification in phosphorylation and reporter assays• Establishing and analysing a Ca2+ (induction) dependent signaling pathway in yeast• Which kinase system activates ABF2 under certain conditions?• How does the signal travel from the plasma membrane localized CBL into the nucleus?• Analysing cipk mutants in terms of ABA and Ca2+ related phenotypes → overlapping
function (e.g. CIPK26)
0 10 20 30 40
CIPK26 + CBL1 + ABF2
ABF2
CIPK26 + CBL1 + ABF2
ABF2
Ca2+ + Ionophore
Ca2+ + DMSO
LUC/GUS
BiFC
YN-CIPK26 + ABF2-YC Autoradiograph
cipk3/cipk26 double mutant needed
1. Kudla et al., 2010 2. Whalley et al., 2011 3. Whalley, 20134. Batistic et al., 2008 5. Yoo et al., 2007
8. Furihata et al., 20067. Umezawa et al., 20138. Zhang et al., 20089. Choi et al., 2005
mean
Fluo
resc
ence
norm
. mea
n Fluo
resc
ence
mean
Fluo
resc
ence
75
55
kDa
ABF2CIPK3.3ABI1PYR8 +ABA
- +
----
ABF2
+++
-
+ ca. 3 µg
ca. 30 ng
ca. 500 ng
ca. 800 ng + 20µMABA
+++
++
A B
sample areainfiltrated area
Cell selection: OFPNucleus focusing: CFPQuantification: YFP
PM-OFPYN - CIPK3.3ABF2 - YCCFPCBL1 - OFP
8. CIPK3 phosphorylates ABF2 in vitro
75
45
55
kDa
ABF2CIPK3.3
+- +
- ABF2
+ ca. 3 µg
ca. 60 ng
45
55- ABF2
Autoradiograph of in vitro phosphorylation assay and respective CBB staining.
ABF2 C1 C3 C4C2 bZIPP P P P
S26/45 S86/94 T135
PCIPK x x x xSnRK2 x x x x xSnRK1 xCDPK x x
ABF2 phosphorylation sites are targets for multiple kinase systems.
Principle of protoplast reporter assay (based on: Ref 5)
0 10 20 30
+ CBL1G2A + CIPK32+Ionophore + Ca
+ CBL1 + CIPK32+Ionophore + Ca
2+Ionophore + Ca
LUC/GUS
ABF2 + RCAR1
Ref 6, 7Ref 8Ref 9
+- +
+
P
Ca2+
Stimulus
CBL1
ABI1
OST1
ABA
ABAABARCARP
Response
ABF2
CIPK3
CIPK3
OST1
ABI1
Nuc
leus
Cyt
opla
sm
Stimulus
ABA
SnRK1CDPK
?
CIPK3
?*
• We identified ABF-type transcription factors as tar-gets of Ca2+ dependent phosphorylation.
• We propose a model that involves CBL dependent CIPK activation at the plasma membrane and subse-quent translocation to the nucleus.
• Combined, our data support activation of ABF2 by CBL1/CIPK3.
• The ABA regulated phosphatase ABI1 dephosphory-lates and inactivates ABF2.
• This suggests that in the absence of stress (ABA) ABI1 keeps ABF2 in an inactive state.
• Upon a stress trigger Ca2+ and ABA can cooperative-ly activate ABF2.
• Together, our data identifies ABF2 as a convergence point of Ca2+ and ABA signaling.
• Combined with published data, we pro-pose a dual deactivation mechanism by ABI1 dephosphorylation of the tar-get (ABF2) as well as the kinase (CIPK/SnRK2).