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Contact information Ca 2+ 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 CBL1 - + Intracellular Ca 2+ 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, Ca 2+ dynamics are associated with the control of gene transcription. Several motifs (including ABA responsive elements - ABRE) have been shown to be Ca 2+ regulated 2,3 . However, the signaling cascades leading to differential gene expres- sion upon Ca 2+ 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 Ca 2+ signaling Kai Edel: E-mail: [email protected] 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 Ca 2+ dependently in protoplasts Calcineurin B like proteins (CBLs) are a Bikonta specific group of Ca 2+ sensor proteins harboring four po- tentially Ca 2+ 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 Ca 2+ 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 Ca 2+ responses 1 . CBL1 CBL1EF2/4 PM 0 5 10 15 * ** Outlook 0 2 4 6 8 2+ Ionophore + ABA+ Ca 2+ Ionophore + Ca 2+ ABA + Ca ABA 2+ Ca control ABF2 + CIPK3.3 + CBL9 LUC/GUS 0 20 40 60 +CBL1EF2/4 2+ Ionophore+Ca +CBL1EF4 2+ Ionophore+Ca +CBL1 2+ Ionophore+Ca CBL1 2+ Ionophore + Ca LUC/GUS ABF2 + RCAR1 + CIPK3.3 6. ABF2 activation by CIPK3/CBL1 requires CBL Ca 2+ 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, Ca 2+ 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 Ca 2+ 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 C4 C2 bZIP P P P P S26 S86/94 T135 S413 ABI5 ATBZIP67 AREB3 ATBZIP12 ABF2 ABF3 ABF4 ABF1 Minimal 35S Luciferase ATG 4x ABRE (ACACGTGTA) -70 Bp 3. Reconstitution of a Ca 2+ 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 extraction Incubation Ca 2+ Ionophore ABA Incubation Transformation LUC GUS 0 20 40 60 +CBL1EF2/4 2+ Ionophore+Ca +CBL1EF4 2+ Ionophore+Ca +CBL1 2+ 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 Ca 2+ dependent signaling pathway in vivo • Study the effects of ABF2 p-site modification in phosphorylation and reporter assays • Establishing and analysing a Ca 2+ (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 Ca 2+ related phenotypes → overlapping function (e.g. CIPK26) 0 10 20 30 40 CIPK26 + CBL1 + ABF2 ABF2 CIPK26 + CBL1 + ABF2 ABF2 Ca 2+ + Ionophore Ca 2+ + 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, 2013 4. Batistic et al., 2008 5. Yoo et al., 2007 8. Furihata et al., 2006 7. Umezawa et al., 2013 8. Zhang et al., 2008 9. Choi et al., 2005 mean Fluorescence norm. mean Fluorescence mean Fluorescence 75 55 kDa ABF2 CIPK3.3 ABI1 PYR8 +ABA - + - - - - ABF2 + + + - + ca. 3 µg ca. 30 ng ca. 500 ng ca. 800 ng + 20µMABA + + + + + A B sample area infiltrated area Cell selection: OFP Nucleus focusing: CFP Quantification: YFP PM-OFP YN - CIPK3.3 ABF2 - YC CFP CBL1 - OFP 8. CIPK3 phosphorylates ABF2 in vitro 75 45 55 kDa ABF2 CIPK3.3 + - + - ABF2 + ca. 3 µg ca. 60 ng 45 55 - ABF2 Autoradiograph of in vitro phosphorylation assay and respective CBB staining. ABF2 C1 C3 C4 C2 bZIP P P P P S26/45 S86/94 T135 P CIPK x x x x SnRK2 x x x x x SnRK1 x CDPK 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 + CIPK3 2+ Ionophore + Ca + CBL1 + CIPK3 2+ Ionophore + Ca 2+ Ionophore + Ca LUC/GUS ABF2 + RCAR1 Ref 6, 7 Ref 8 Ref 9 + - + + P Ca 2+ Stimulus CBL1 ABI1 OST1 ABA ABA ABA RCAR P Response ABF2 CIPK3 CIPK3 OST1 ABI1 Nucleus Cytoplasm Stimulus ABA SnRK1 CDPK ? CIPK3 ? * • We identified ABF-type transcription factors as tar- gets of Ca 2+ 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 Ca 2+ and ABA can cooperative- ly activate ABF2. • Together, our data identifies ABF2 as a convergence point of Ca 2+ 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).
