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Sucrose-induced Receptor Kinase 1 is Modulated by an Interacting Kinase with Short Extracellular Domain*

2019; Elsevier BV; Volume: 18; Issue: 8 Linguagem: Inglês

10.1074/mcp.ra119.001336

ISSN

1535-9484

Autores

Xu Wu, Liang‐Cui Chu, Lin Xi, Heidi Pertl-Obermeyer, Zhi Li, Kamil Skłodowski, Clara Sánchez‐Rodríguez, Gerhard Obermeyer, Waltraud X. Schulze,

Tópico(s)

Ion Transport and Channel Regulation

Resumo

Sucrose as a product of photosynthesis is the major carbohydrate translocated from photosynthetic leaves to growing nonphotosynthetic organs such as roots and seeds. These growing tissues, besides carbohydrate supply, require uptake of water through aquaporins to enhance cell expansion during growth. Previous work revealed Sucrose Induced Receptor Kinase, SIRK1, to control aquaporin activity via phosphorylation in response to external sucrose stimulation. Here, we present the regulatory role of AT3G02880 (QSK1), a receptor kinase with a short external domain, in modulation of SIRK1 activity. Our results suggest that SIRK1 autophosphorylates at Ser-744 after sucrose treatment. Autophosphorylated SIRK1 then interacts with and transphosphorylates QSK1 and QSK2. Upon interaction with QSK1, SIRK1 phosphorylates aquaporins at their regulatory C-terminal phosphorylation sites. Consequently, in root protoplast swelling assays, the qsk1qsk2 mutant showed reduced water influx rates under iso-osmotic sucrose stimulation, confirming an involvement in the same signaling pathway as the receptor kinase SIRK1. Large-scale phosphoproteomics comparing single mutant sirk1, qsk1, and double mutant sirk1 qsk1 revealed that aquaporins were regulated by phosphorylation depending on an activated receptor kinase complex of SIRK1, as well as QSK1. QSK1 thereby acts as a coreceptor stabilizing and enhancing SIRK1 activity and recruiting substrate proteins, such as aquaporins. Sucrose as a product of photosynthesis is the major carbohydrate translocated from photosynthetic leaves to growing nonphotosynthetic organs such as roots and seeds. These growing tissues, besides carbohydrate supply, require uptake of water through aquaporins to enhance cell expansion during growth. Previous work revealed Sucrose Induced Receptor Kinase, SIRK1, to control aquaporin activity via phosphorylation in response to external sucrose stimulation. Here, we present the regulatory role of AT3G02880 (QSK1), a receptor kinase with a short external domain, in modulation of SIRK1 activity. Our results suggest that SIRK1 autophosphorylates at Ser-744 after sucrose treatment. Autophosphorylated SIRK1 then interacts with and transphosphorylates QSK1 and QSK2. Upon interaction with QSK1, SIRK1 phosphorylates aquaporins at their regulatory C-terminal phosphorylation sites. Consequently, in root protoplast swelling assays, the qsk1qsk2 mutant showed reduced water influx rates under iso-osmotic sucrose stimulation, confirming an involvement in the same signaling pathway as the receptor kinase SIRK1. Large-scale phosphoproteomics comparing single mutant sirk1, qsk1, and double mutant sirk1 qsk1 revealed that aquaporins were regulated by phosphorylation depending on an activated receptor kinase complex of SIRK1, as well as QSK1. QSK1 thereby acts as a coreceptor stabilizing and enhancing SIRK1 activity and recruiting substrate proteins, such as aquaporins. Growth and development of a plant require precise control of carbon assimilation, transport and storage (1Chapin F.S. Schulze E.D. Mooney H.A. The ecology and economics of storage in plants.Annu. Rev. Ecol. Syst. 1990; 21: 423-447Crossref Scopus (1346) Google Scholar). In this context, sucrose as a main product of photosynthesis in most plant species is the major carbohydrate translocated within the phloem to serve as carbon supply for nonphotosynthetic tissues such as roots or seeds. Sucrose is used for the maintenance of cellular metabolism, as precursor for cell wall biosynthesis, and a major storage sugar in vacuoles. Mechanisms of how sucrose is loaded into the phloem (2Sauer N. Stolz J. SUC1 and SUC2: Two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein.Plant J. 1994; 6: 67-77Crossref PubMed Scopus (298) Google Scholar, 3Kühn C. Franceschi V.R. Schulz A. Lemoine R. Frommer W.B. Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements.Science. 1997; 275: 1298-1300Crossref PubMed Scopus (382) Google Scholar) and distributed within the plant (4Weber H. Borisjuk L. Heim U. Sauer N. Wobus U. A role for sugar transporters during seed development: Molecular characterization of a hexose and a sucrose carrier in fava bean seeds.Plant Cell. 1997; 9: 895-908Crossref PubMed Scopus (218) Google Scholar) are well understood and were completed with discovery and characterization of sucrose-exporting SWEET family (5Chen L.Q. Qu X.Q. Hou B.H. Sosso D. Osorio S. Fernie A.R. Frommer W.B. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.Science. 2011; 35: 207-211Google Scholar). Besides sucrose, expansion of cells during growth and storage requires the influx of water. Since the discovery of aquaporins as water channels within membranes (6Javot H. Lauvergeat V. Santoni V. Martin-Laurent F. Guclu J. Vinh J. Heyes J. Franck K.I. Schaffner A.R. Bouchez D. Maurel C. Role of a single aquaporin isoform in root water uptake.Plant Cell. 2003; 15: 509-522Crossref PubMed Scopus (285) Google Scholar), their regulation through C-terminal phosphorylation was unraveled (7Maurel C. Kado R.T. Guern J. Chrispeels M.J. Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.EMBO J. 1995; 14: 3028-3035Crossref PubMed Scopus (273) Google Scholar, 8Prak S. Hem S. Boudet J. Viennois G. Sommerer N. Rossignol M. Maurel C. Santoni V. Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: role in subcellular trafficking of AtPIP2;1 in response to salt stress.Mol. Cell. Proteomics. 2008; 7: 1019-1030Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 9di Pietro M. Vialaret J. Li G.W. Hem S. Prado K. Rossignol M. Maurel C. Santoni V. Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots.Mol. Cell. Proteomics. 2013; 12: 3886-3897Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Aquaporins play important roles during lateral root growth (10Péret B. Li G. Zhao J. Band L.R. Voβ U. Postaire O. Luu D.T. Da Ines O. Casimiro I. Lucas M. Wells D.M. Lazzerini L. Nacry P. King J.R. Jensen O.E. Schäffner A.R. Maurel C. Bennett M.J. Auxin regulates aquaporin function to facilitate lateral root emergence.Nat. Cell Biol. 2012; 14: 991-998Crossref PubMed Scopus (261) Google Scholar, 11Vandeleur R.K. Sullivan W. Athman A. Jordans C. Gilliham M. Kaiser B.N. Tyerman S.D. Rapid shoot-to-root signalling regulates root hydraulic conductivity via aquaporins.Plant Cell Environ. 2014; 37: 520-538Crossref PubMed Scopus (120) Google Scholar) and seed development (12Maurel C. Chrispeels M. Lurin C. Tacnet F. Geelen D. Ripoche P. Guern J. Function and regulation of seed aquaporins.J. Exp. Bot. 1997; 48: 421-430Crossref PubMed Google Scholar). Arabidopsis contains ∼600 receptor like kinases which play critical roles in regulation of general signal perception and transduction as well as plant growth and defense (13Zulawski M. Schulze G. Braginets R. Hartmann S. Schulze W.X. The Arabidopsis Kinome: Phylogeny and evolutionary insights into functional diversification.BMC Genomics. 2014; 15: 548Crossref PubMed Scopus (78) Google Scholar). There are about 223 LRR receptor-like kinases in Arabidopsis (14Shiu S.-H. Bleecker A.B. Plant receptor-like kinase gene family: Diversity, function, and signaling.Sci. STKE. 2001; 113: 1-13Google Scholar), and only about 60 of these have been functionally characterized (15Wu Y. Xun Q. Guo Y. Zhang J. Cheng K. Shi T. He K. Hou S. Gou X. Li J. Genome-wide expression pattern analyses of the Arabidopsis leucine-rich repeat receptor-like kinases.Mol Plant. 2016; 9: 289-300Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Receptor kinases with a large extracellular domain are considered to play key roles in ligand binding and perception, being specific to a single signaling pathways (16Shinohara H. Mori A. Yasue N. Sumida K. Matsubayashi Y. Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 3897-3902Crossref PubMed Scopus (112) Google Scholar). In contrast, receptor kinases with short extracellular domains are often found to be involved in more than one signaling pathway and have coreceptor functions. For example, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATION RECEPTOR KINASE 1 (BAK1, also known as SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3, SERK3) function as brassinosteroid (BR) 1The abbreviations used are:BRbrassinosteroidSIRK 1sucrose-induced receptor kinaseFWHMfull width half maximumFDRfalse discovery rates. 1The abbreviations used are:BRbrassinosteroidSIRK 1sucrose-induced receptor kinaseFWHMfull width half maximumFDRfalse discovery rates. receptor and coreceptor, respectively. In the BR signaling pathway, BR binding induces a basal activation of the receptor BRI1 for binding BAK1, and transactivation occurs between BRI1 and BAK1 to fully activate BRI1 to enhance the phosphorylation of downstream substrate (17Li J. Wen J. Lease K.A. Doke J.T. Tax F.E. Walker J.C. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling.Cell. 2002; 110: 213-222Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar, 18Nam K.H. Li J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling.Cell. 2002; 110: 203-212Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar, 19Wang G. Wu W.W. Zeng W. Chou C.L. Shen R.F. Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.J. Proteome Res. 2006; 5: 1214-1223Crossref PubMed Scopus (229) Google Scholar, 20Wang X. Goshe M.B. Sonderblom E.J. Phinney B.S. Kuchar J.A. Li J. Asami T. Yoshida S. Huber S.C. Clouse S.D. Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis Brassinosteroid-insensitive 1 receptor kinase.Plant Cell. 2005; 17: 1685-1703Crossref PubMed Scopus (316) Google Scholar, 21Wang X. Kota U. He K. Blackburn K. Li J. Goshe M.B. Huber S.C. Clouse S.D. Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling.Dev. Cell. 2008; 15: 220-235Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 22Russinova E. Borst J.W. Kwaaitaal M. Cano-Delgado A. Yin Y. Chory J. De Vries S.C. Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1).Plant Cell. 2004; 16: 3216-3229Crossref PubMed Scopus (371) Google Scholar, 23Yun H.S. Bae Y.H. Lee Y.J. Chang S.C. Kim S.K. Li J. Nam K.H. Analysis of phosphorylation of the BRI1/BAK1 complex in arabidopsis reveals amino acid residues critical for receptor formation and activation of BR signaling.Mol. Cells. 2009; 27: 183-190Crossref PubMed Scopus (42) Google Scholar, 24Santiago J. Henzler C. Hothorn M. Molecular mechanism for plant steroid receptor activation by somatic embryogenesis co-receptor kinases.Science. 2013; 341: 889-892Crossref PubMed Scopus (246) Google Scholar). BAK1 is also coreceptor recruited to receptor kinase FLAGELLIN-SENSING 2 (FLS2) after perception of the flagellin peptide (flg22). The formation of a complex of receptor FLS2, coreceptor BAK1 and ligand flg22 leads to a full activation of downstream immune defense signaling (25Roux M. Schwessinger B. Albrecht C. Chinchilla D. Jones A. Holton N. Malinovsky F.G. Tor M. de Vries S. Zipfel C. The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens.Plant Cell. 2011; 23: 2440-2455Crossref PubMed Scopus (469) Google Scholar, 26Heese A. Hann D.R. Gimenez-Ibanez S. Gajdanowicz P. Jones A.M. He K. Li J. Schroeder J.I. Peck S.C. Rathjen J. The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12217-12222Crossref PubMed Scopus (787) Google Scholar, 27Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nurnberger T. Jones J.D. Felix G. Boller T. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defense.Nature. 2007; 448: 497-500Crossref PubMed Scopus (1274) Google Scholar, 28Sun Y. Li L. Macho A.P. Han Z. Hu Z. Zipfel C. Zhou J.M. Chai J. Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex.Science. 2013; 342: 624-628Crossref PubMed Scopus (458) Google Scholar). In addition, SERKs including BAK1 function as coreceptors of IDA-receptors HAE/HLS2 and EPF-receptor ERECTA in regulation of floral organ abscission and stomatal patterning (29Meng X. Chen X. Mang H. Liu C. Yu X. Gao X. Torii K.U. He P. Shan L. Differential Function of Arabidopsis SERK Family Receptor-like Kinases in Stomatal Patterning.Curr. Biol. 2015; 25: 2361-2372Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 30Meng X. Zhou J. Tang J. Li B. de Oliveira M.V. Chai J. He P. Shan L. Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis.Cell Reports. 2016; 14: 1330-1338Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), as well as in phytosulfokine signaling (31Wang J. Li H. Han Z. Zhang H. Wang T. Lin G. Chang J. Yang W. Chai J. Allosteric receptor activation by the plant peptide hormone phytosulfokine.Nature. 2015; 525: 265-268Crossref PubMed Scopus (151) Google Scholar) and other pathways. brassinosteroid sucrose-induced receptor kinase full width half maximum false discovery rates. brassinosteroid sucrose-induced receptor kinase full width half maximum false discovery rates. Recently, several receptor kinases were shown to directly interact with and regulate plasma membrane transmembrane transporters, channels and proton pumps. For example, different LRR-receptor kinases, besides linking to cytoplasmic signaling cascades, directly regulate the plasma membrane H+-ATPases (32Haruta M. Sabat G. Stecker K. Minkoff B.B. Sussman M.R. A peptide hormone and its receptor protein kinase regulates plant cell expansion.Science. 2014; 343: 408-411Crossref PubMed Scopus (467) Google Scholar, 33Caesar K. Elgass K. Chen Z. Huppenberger P. Witthöft J. Schleifenbaum F. Blatt M.R. Oecking C. Harter K. A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana.Plant J. 2011; 66: 528-540Crossref PubMed Scopus (80) Google Scholar, 34Fuglsang A.T. Kristensen A. Cuin T. Schulze W.X. Persson J. Thuesen K.H. Ytting C.K. Oehlschlaeger C. Mahmood K. Sondergaard T.E. Shabala S. Palmgren M.G. Receptor kinase mediated control of primary active proton pumping at the plasma membrane.Plant J. 2014; 80: 951-964Crossref PubMed Scopus (87) Google Scholar), Ca2+-ATPases (35Frei dit Frey N. Mbengue M. Kwaaitaal M. Nitsch L. Altenbach D. Haweker H. Lozano-Duran R. Njo M.F. Beeckman T. Huettel B. Borst J.W. Panstruga R. Robatzek S. Plasma membrane calcium ATPases are important components of receptor-mediated signaling in plant immune responses and development.Plant Physiol. 2012; 159: 798-809Crossref PubMed Scopus (96) Google Scholar) or aquaporins (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The recent discovery of such short, direct regulatory circuits within the plasma membrane between receptor kinases and transporters or channels suggests that this is a generic modular principle allowing plants to very rapidly adjust to changing environments. A prerequisite for the function of such short signaling circuits are highly dynamic protein-protein interactions between kinases and the respective transporters or channels. Based on existing evidence from ABA-induced membrane protein complex dynamics in membrane nanodomains (37Demir F. Hontrich C. Blachutzik J.O. Scherzer S. Reinders Y. Kierszniowska S. Schulze W.X. Harms G.S. Hedrich R. Geiger D. Kreuzer I. Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 8296-8301Crossref PubMed Scopus (153) Google Scholar), also lateral segregation within the membrane may play a key role in the dynamics of such regulatory interactions. In previous research (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), we found that Sucrose-induced receptor kinase SIRK1 (AT5G10020) can regulate aquaporins through phosphorylation under conditions of external sucrose supply. Upon sucrose stimulation, SIRK1 was found to form a complex with another yet uncharacterized receptor kinase with a short extracellular domain (AT3G02880). Based on our findings in this work we here name this protein QSK1, qiān shŏu, (Chinese: 千 手) "thousand hands" kinase. Interestingly, the localization of QSK1 within the plasma membrane between detergent resistant membrane fractions (DRM) and detergent soluble fractions (DSM) was previously found to be highly dependent on cytoskeleton integrity (38Szymanski W.G. Zauber H. Erban A. Wu X.N. Schulze W.X. Cytoskeletal components define protein location to membrane microdomains.Mol. Cell. Proteomics. 2015; 14: 2493-2509Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Disruption of actin filaments resulted in a depletion of QSK1 from DRM and increase in DSM location which was visible by enhanced appearance of the punctate location of QSK1 (39Schlesier B. Breton F. Mock H.P. A hydroponic culture system for growing Arabidopsis thaliana plantlets under sterile conditions.Plant Mol. Biol. Reporter. 2003; 21: 449-456Crossref Scopus (46) Google Scholar). In contrast, disruption of microtubules resulted in a more uniform and less structured localization and was accompanied by general internalization processes or reduced secretion (38Szymanski W.G. Zauber H. Erban A. Wu X.N. Schulze W.X. Cytoskeletal components define protein location to membrane microdomains.Mol. Cell. Proteomics. 2015; 14: 2493-2509Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, the role of QSK1 in regulation of receptor kinase SIRK1 and aquaporins remained to be elucidated. In the present study, we now explore the activation mechanism of SIRK1 and QSK1 using aquaporins as a known phosphorylation and activation target of the SIRK1/QSK1 complex. We conclude that QSK1 functions in stabilizing SIRK1 activity like a coreceptor in other receptor kinase signaling pathways. To investigate the role of QSK1 (At3g02880) and QSK2 (At5g16590) in the regulation of aquaporins, we performed different experiments involving mass-spectrometry based proteomic analyses. First, a comparative phosphoproteomic analysis of wild type, qsk1 mutant, sirk1 mutant, and sirk1 qsk1 was performed under sucrose starvation and sucrose resupplied conditions. Secondly, pull-down experiments were carried out using SIRK1-GFP or QSK1-GFP as bait proteins. Furthermore, in vitro phosphorylation assays were done using purified recombinant QSK1 and SIRK1 kinase domains and synthetic peptides as phosphorylation targets. All these experiments were carried out with at least three biological replicates per genotype or treatment and results are presented as averages from these biological replicates. We used the MaxQuant/Perseus data analysis platform for quantitative analysis in all proteomics experiments. Arabidopsis seeds of wild type (col-0), sirk1 single mutant (SALK_125543), qsk1 single mutant (SALK_019840), qsk2 single mutant (WiscDsLoxHs082_03E), sirk1 qsk1 double mutant (crossing of the sirk1 and qsk1 T-DNA insertion lines), sirk1 qsk1 qsk2 triple mutant (crossing of sirk1, qsk1, and qsk2 T-DNA insertional lines), as well as overexpression lines 35S::SIRK1-GFP and 35S::QSK1-GFP were used. Homozygous T-DNA insertional mutants sirk1, qsk1 and double mutant sirk1 qsk1 were confirmed via PCR amplification using T-DNA border primer LBb1.3 (5′-ATTTTGCCGATTTCGGAAC-3′) and gene-specific primers (SIRK1-RP: 5′-TTTCCAGCATTTCCAACACTC-3′, SIRK1-LP: 5′-CACTAAGCTTGTTGAGGTCGC-3′; QSK1-RP: 5′-CAAACCAGGTCCATCAAGATC-3′, QSK1-LP: 5′-GAGATTCCGTCGCTTCTCTTC-3′). Lack of SIRK1 gene and protein expression was already characterized previously for the sirk1 mutant (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and Lack of QSK1 expression was demonstrated for the qsk1 mutant (supplemental Fig. S1). Mutants of qsk2 and qsk1 qsk2 were confirmed via PCR amplification using T-DNA border primer LB (5′-TGATCCATGTAGATTTCCCGGACATGAAG-3) and gene specific primer (QSK2-RP: 5′-TTCCATTCACTGCAGTCTGC-3′, QSK2-LP: 5′-GCAGAAGCTTTCAGCAATCC-3′). Plants were germinated and grown under 16/8 day/night (22 °C, 120 μE/s*m2) in ½ MS medium plus 0.5% sucrose in a hydroponic cultivation system (39Schlesier B. Breton F. Mock H.P. A hydroponic culture system for growing Arabidopsis thaliana plantlets under sterile conditions.Plant Mol. Biol. Reporter. 2003; 21: 449-456Crossref Scopus (46) Google Scholar). After 19 days, seedlings were starved by changing the growth medium to a sucrose-free medium and leaving the culture vessels in the dark for 48 h as described (40Niittylä T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Sucrose was then resupplied to a final concentration of 30 mm for 3 min before roots were harvested for microsomal protein preparation. Total RNA was extracted from the qsk1 plants using the Plant RNA Mini Kit (peQlab, Germany) according to the instructions provided by the manufacturer. RNA was digested with DNaseI (Roche Diagnostics, Germany) to remove the genomic DNA. First-strand cDNA was synthesized using PrimeScript RT reagent kit (TaKaRa). Quantitative real-time PCR analysis was performed using the Bio-Rad CFX Connect real-time PCR system (BioRad Laboratories; Munich, Germany) with the SYBR green detection protocol (TaKaRa, Saint-Germain-en-Laye, France). The Actin and Tubulin genes were used as reference genes, and the relative expression of the gene of interest was calculated by the 2∧-ΔΔCq method. RT Primers for QSK1 was F 5-TGAGTCATGCCAATCTCGTGAC-3, R 5-GCAATATCGCAGACAAGCTTCC-3. Primers used for Actin and Tubulin were: Actin-F 5-ACTTTCATCAGCCGTTTTGA-3, Actin-R-5-ACGATTGGTTGAATATCATCAG-3 and Tubulin-F 5-ACCTACTGGTCTGAAGATGGCAT-3 and Tubulin-R5-TTTCTCCTGAACATAGCTGTGAAC-3. For ratiometric bimolecular Fluorescence Complementation (rBiFC) of Arabidopsis proteins, cDNAs of the following genes were cloned into rBiFC plasmids (41Grefen C. Blatt M.R. A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC).BioTechniques. 2012; 53: 311-314Crossref PubMed Scopus (104) Google Scholar): SIRK1, or SIRK1 with phosphorylation site mutations SIRK1S744A, SIRK1S744D, QSK1, QSK1 with phosphorylation site mutations QSK1S621AS626A, QSK1S621DS626D, QSK2 and PIP2;4. SIRK1, SIRK1S744A and SIRK1S744D were cloned as fusions with the C-terminal half of YFP, whereas QSK1, QSK1S621AS626A, QSK1S621DS626D, QSK2 and PIP2;4 were cloned as fusions with the N-terminal half of YFP. All constructs were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Positive colonies were confirmed by spectinomycin and rifampicin resistance and colony PCR. To produce overexpression lines of SIRK1 and QSK1 for pull-down analysis (35S::SIRK1-GFP and 35S::QSK1-GFP), cDNA of the SIRK1 gene and QSK1 gene without a stop code were cloned into the plant transformation vector pEZR(H)-LN (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and fused with GFP coding sequence. Homozygous T2 transgenic lines were further selected by means of segregation analysis, and T3 seeds were used for experiments. The fluorescence of GFP in the transgenic lines was checked using confocal laser-scanning microscopy (TCS SP5, Leica Microsystems CMS GmbH, Wetzlar, Germany). For purification of the cytoplasmic domain of SIRK1 protein (SIRK1-CD, amino acid 625–1048) and QSK1 protein (QSK1-CD, amino acid 276–627) to be used in in vitro kinase assays, SIRK1-CD and QSK1-CD were cloned into Escherichia coli BL21(DE3) expressing plasmid pETGST 1a and fused with His and GST tags, resulting in the plasmid His-GST-SIRK1-CD and His-GST-QSK1-CD. Plasmids His-GST-SIRK1-CD and His-GST-QSK1-CD were transformed into Escherichia coli BL21 (DE3). After 5 h induction by IPTG (isopropyl β-d-thiogalactopyranoside), cells were harvested and lysed using BugBuster Protein Extraction Reagent (Novagen, Nottingham, UK), soluble fractions were used over gravity flow Ni2+-NTA Sepharose columns (1 ml, IBA GmbH, Goettingen, Germany) for His-GST-SIRK1-CD and His-GST-QSK1-CD protein purification. A total of 1 to 1.5 g of roots (fresh weight) was homogenized in 10 ml ice-cold extraction buffer (330 mm mannitol, 100 mm KCl, 1 mm EDTA, 50 mm Tris-MES, fresh 5 mm DTT, and 1 mm phenylmethylsulfonylfluoride, pH 7.5) (42Pertl H. Himly M. Gehwolf R. Kriechbaumer R. Strasser D. Michalke W. Richter K. Ferreira F. Obermeyer G. Molecular and physiological characterisation of a 14–3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth.Planta. 2001; 213: 132-141Crossref PubMed Scopus (50) Google Scholar) in the presence of 0.5% v/v proteinase inhibitor mixture (Sigma-Aldrich, Taufkirchen, Germany) and phosphatase inhibitors (25 mm NaF, 1 mm Na3VO4, 1 mm benzamidin, 3 μm proteinase inhibitor leupeptin). The homogenate was centrifuged for 15 min at 7500 × g at 4 °C. The pellet was discarded, and the supernatant was centrifuged for 75 min at 48,000 × g at 4 °C. The microsomal pellet was resuspended in 100 μl of membrane buffer (330 mm mannitol, 25 mm Tris-MES, 0.5 mm DTT) or UTU (6 m urea, 2 m thiourea, pH 8). Further tryptic digestion, desalting over C18 and enrichment of phosphopeptides over titanium dioxide beads was performed as described (43Wu X.N. Schulze W.X. Phosphopeptide profiling of receptor kinase mutants.Methods Mol. Biol. 2015; 1306: 71-79Crossref PubMed Scopus (6) Google Scholar). Root microsomal proteins (100 μg) isolated as described above was incubated with 25 μl of anti-GFP agarose beads (Chromotek, Planegg, Germany) for two hours on a rotating wheel at 4 °C (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). After incubation, the beads were collected by centrifugation and washed two times with 500 μl wash buffer (10 mm Tris/HCl pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 0.01% IGEPAL). For protein-protein interaction assays, the proteins were eluted from the beads with 100 μl UTU (6 m urea, 2 m thiourea), pH 8, before in-solution tryptic digestion. For kinase activity assays, three more washing steps were carried out with one-time wash buffer (10 mm Tris/HCl, pH 7.5, 300 mm NaCl, 0.5 mm EDTA) and two times kinase reaction buffer (40 mm Tris/HCl pH 7.5, 10 mm MgCl2, 0.1% BSA, 2 mm DTT). SIRK1-GFP and QSK1-GFP fusion proteins were affinity purified over anti-GFP beads (see above). A luciferase-based kinase activity assay was performed as described (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The agarose beads with GFP-tagged proteins were re-suspended into 30 μl kinase reaction buffer with ATP and the generic kinase substrate myelin basic protein (40 mm Tris/HCl pH 7.5, 10 mm MgCl2, 0.1% BSA, 2 mm DTT, 100 μm ATP, 0.4 μg/μl myelin basic protein). After incubation for one hour, 30 μl ADP-GLO Reagents (Promega, Mannheim, Germany) was added and incubated for 40 min. Then Kinase Detection Reagents were added and incubated for another hour. Luminescence as a measure of ATP conversion from ADP was recorded with a luminometer (Tecan M200 Pro, Crailsheim, Germany). In general, protein isolations from three independent batches of roots were tested, and the average activity value is presented. Kinase activity assays were performed as described above, except that 10 pmol of the peptides FSDQPVMLDVYSPDR (SIRK1), LIEEVSHSSGSPNPVSD (QSK1), or ALGSFGSFGSFR (PIP2;4) were used as a substrate and His-GST-SIRK1-CD and His-GST-QSK1-CD protein were used as kinases. All peptides were synthesized based on experimentally identified phosphopeptides in this work or previous studies (36Wu X. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.Mol. Cell. Proteomics. 2

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