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The Sensor Histidine Kinases AHK2 and AHK3 Proceed into Multiple Serine/Threonine/Tyrosine Phosphorylation Pathways in Arabidopsis thaliana

2015; Elsevier BV; Volume: 9; Issue: 1 Linguagem: Inglês

10.1016/j.molp.2015.10.002

1674-2052

Rebecca Dautel, Xu Wu, Michael Heunemann, Waltraud X. Schulze, Klaus Harter,

Plant Gene Expression Analysis

Cytokinins are N6-substituted adenine derivatives that are involved in the regulation of numerous aspects of plant growth and development. These include the control of cell division, leaf senescence, apical dominance, sink/source relationship, vascular and embryonic development, and apical meristem activity (Kieber and Schaller, 2014Kieber J.J. Schaller G.E. Cytokinins.Arabidopsis Book. 2014; 12: e0168Crossref Google Scholar). In addition, there is increasing evidence for the function of cytokinins in abiotic stress responses (Zwack and Rashotte, 2015Zwack P.J. Rashotte A.M. Interactions between cytokinin signaling and abiotic stress responses.J. Exp. Bot. 2015; 66: 4863-4871Crossref PubMed Scopus (203) Google Scholar). The cytokinin signal transduction pathway in plants is considered to be a canonical two-component, multistep phosphorelay system (TCS). Upon cytokinin binding, the sensor histidine kinases (HK) autophosphorylate at a conserved His residue. The phosphoryl group is then transferred to a conserved Asp residue in the HK's own receiver domain. The His-containing phosphotransfer proteins (HP) perceive the phosphoryl group from the receiver domain of the HKs and transfer it to the Asp in the receiver of response regulators (RR). The RRs function preferentially either as transcription factors mediating cytokinin-regulated gene expression (B-type RRs) or as negative regulators of cytokinin signaling (A-type RRs). The cytokinin sensor HK family of Arabidopsis consists of three members: AHK2, AHK3, and AHK4. They have overlapping functions in cytokinin responses, are localized as dimers in the ER membrane, and bind cytokinin via their CHASE domain (Kieber and Schaller, 2014Kieber J.J. Schaller G.E. Cytokinins.Arabidopsis Book. 2014; 12: e0168Crossref Google Scholar). To date, there is no evidence that AHK2, -3, and -4 and the TCS elements acting downstream of the HKs, namely the Arabidopsis HPs and RRs (ARR), have properties not related to a canonical His-to-Asp phosphorelay. Interestingly, the osmolarity sensing pathway in yeast has been shown to be a canonical TCS phosphorelay, which eventually activates the Hog1 mitogen-activated protein kinase (MAPK) pathway upon high-osmolarity stress (Posas and Saito, 1998Posas F. Saito H. Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator.EMBO J. 1998; 17: 1385-1394Crossref PubMed Scopus (250) Google Scholar). Having this signaling transition from a TCS to a classical MAPK pathway in mind, we sought to test whether similar mechanisms may also exist in plants regarding cytokinin signaling. To study the potential of the HK-dependent TCS phosphorelay to cause Ser/Thr/Tyr modification, comparative phosphoproteomics experiments in the absence or presence of the cytokinin were carried out with seedlings of the wild type (WT; Col-0) and the ahk2ahk3 double mutant, which displays a relatively weak cytokinin hyposensitive phenotype (Higuchi et al., 2004Higuchi M. Pischke M.S. Mähönen A.P. Miyawaki K. Hashimoto Y. Seki M. Kobayashi M. Shinozaki K. Kato T. Tabata S. et al.In planta functions of the Arabidopsis cytokinin receptor family.Proc. Natl. Acad. Sci. USA. 2004; 101: 8821-8826Crossref PubMed Scopus (532) Google Scholar). Seedlings were grown hydroponically for a reciprocal metabolic 15N-labeling approach under continuous white light for 14 days in media without cytokinin. Then the seedlings were either exposed to the final concentration of 100 ng/ml kinetin, a functional cytokinin, for 10 min or mock-treated before harvesting. The experiments were carried out in four biological replicates for each combination of 14N/15N labeling, and the data were averaged between the replicates. We chose this early time point to identify phosphorylation/dephosphorylation events before the onset of major cytokinin-controlled gene expression and protein accumulation. Protein extraction, tryptic digestion, and phosphopeptide enrichment were carried out according to the Supplemental Information. Tryptic peptides were analyzed by liquid chromatography–tandem mass spectrometry using a Quadrupole-Orbitrap hybrid mass spectrometer. Proteins were identified by tandem mass spectrometry using information-dependent acquisition of fragmentation spectra of multiple charged peptides. Acquired spectra were matched against the Arabidopsis proteome. For quantitation, ratios between heavy (15N) and light (14N) forms of each peptide were calculated. Ratios from reciprocal experiments were converted to mutant-versus-WT ratios. Proteins significantly up- or downregulated in their phosphorylation pattern were defined by pairwise t-testing, if peptides were identified in both replica experiments. For peptides identified only in one replica experiment, a two-fold-change cutoff was applied. In total, 836 phosphopeptides were identified in at least one genotype or condition (Supplemental Tables 1 and 2). Of these, 553 phosphopeptides upon kinetin treatment and 358 phosphopeptides under mock conditions were quantified and are presented as ahk2ahk3/WT ratio. In mock-treated seedlings, only very few significant differences in the phosphoproteome of the WT and ahk2ahk3 seedlings were detectable (34 differentially phosphorylated proteins; Figure 1A and Supplemental Tables 1 and 2). In contrast, 150 phosphopeptides were found to be differentially modified between the WT and ahk2ahk3 seedlings after kinetin treatment. There was an overlap of 12 phosphopeptides between mock- and kinetin-treated samples (Supplemental Table 3; the representative spectra for one of the overlapping phosphopeptides, LIEEVSHSSG(pS)PNPVSD, are exemplarily provided in Supplemental Figure 1). The differences of phosphorylation level in the ahk2ahk3 double mutant compared with the WT were much more pronounced when plants were treated with kinetin (Supplemental Figure 2). Thus, there are major changes in the Ser/Thr/Tyr phosphoproteome that are dependent on the presence of AHK2 and AHK3, and these changes become particularly apparent in the presence of kinetin (Supplemental Figure 2). We therefore focused our further analysis on the dataset obtained from the kinetin-treated seedlings (hereafter called the kinetin dataset). Of the 150 differentially modified phosphopeptides detected in the kinetin dataset, 72 displayed a reduced (log2 ahk2ahk3/wt ≤ −1) and 78 phosphopeptides an increased (log2 ahk2ahk3/wt ≥ 1) phosphorylation in the ahk2ahk3 mutant compared with the WT. Given that almost equal numbers of proteins were found with increased and decreased phosphorylation, a large number of modification changes appear to be caused through changes in phosphatase activities. The 150 phosphopeptides contained 157 Ser, 59 Thr, and 23 Tyr sites. Using Motif-X and the standard settings of occurrence of a least 10 and a significance threshold of 10−6, no significant phosphorylation motifs were found, not even for the 157 Ser sites. For the Thr and Tyr sites the dataset is too small to allow efficient motif identification, even by using a lower significance threshold. The differentially phosphorylated proteins have predicted or experimentally determined localization to the plasma membrane, ER, cytosol, nucleus, or extracellular space. Their functional categorization using Mapman demonstrates that the AHK2/AHK3-dependent TCS phosphorelay influences the modification state of many proteins (Figure 1A), particularly calcium signaling, cellular organization and division, and regulation of transcription (Figure 1A). Interestingly, among the phosphopeptides differentially modified between ahk2ahk3 double mutant and WT in the kinetin data set of WT seedlings we found AHK2 itself, which was phosphorylated at Thr4, a site previously identified in two other phosphoproteomics studies (Sugiyama et al., 2008Sugiyama N. Nakagami H. Mochida K. Daudi A. Tomita M. Shirasu K. Ishihama Y. Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis.Mol. Syst. Biol. 2008; 4: e1-7Crossref Scopus (315) Google Scholar, Nakagami et al., 2010Nakagami H. Sugiyama N. Mochida K. Daudi A. Yoshida Y. Toyoda T. Tomita M. Ishihama Y. Shirasu K. Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants.Plant Physiol. 2010; 153: 1161-1174Crossref PubMed Scopus (308) Google Scholar), and at Ser596, very close to the His kinase domain of AHK2 (Figure 1B and 1F; Supplemental Figure 3). Both previous studies identified another AHK2 phosphorylation site at Thr740 located within the HATPase domain (Figure 1F). The AHK2 phosphorylation sites identified here were found in phosphorylated form in the kinetin-treated WT but not in the mock-treated WT datasets. ARR7, a canonical A-type ARR, shows increased phosphorylation within a predicted phosphorylation hotspot (Christian et al., 2012Christian J.O. Braginets R. Schulze W.X. Walther D. Characterization and prediction of protein phosphorylation hotspots in Arabidopsis thaliana.Front. Plant Sci. 2012; 3: 207Crossref PubMed Scopus (19) Google Scholar) in the ahk2ahk3 mutant in the kinetin dataset at two adjacent sites (Ser197 and Ser199) in the C terminus (Figure 1B and 1F). ARR7 is a negative regulator of cytokinin signaling, and functions together with ARR15 in the cytokinin-controlled maintenance of the stem cell systems embedded in shoot and root meristems (Zhao et al., 2010Zhao Z. Andersen S.U. Ljung K. Dolezal K. Miotk A. Schultheiss S.J. Lohmann J.U. Hormonal control of the shoot stem-cell niche.Nature. 2010; 465: 1089-1092Crossref PubMed Scopus (349) Google Scholar). It is, therefore, tempting to speculate that the phosphorylation of AHK2 and dephosphorylation of ARR7 modulate the sensitivity state of the TCS phosphorelay, thereby influencing stem cell maintenance and also other cytokinin-related processes. Another protein with increased phosphorylation in ahk2ahk3 in the kinetin dataset is cytokinin oxidase/dehydrogenase 2 (CKX2) (Figure 1B), which is predicted to be located in the ER lumen. CKX2 is a member of a multiprotein family, which catalyzes the breakdown of cytokinins. CKX2 phosphorylation may modulate its enzymatic activity and, thereby, the amount of active cytokinin in the ER lumen. Consequently, this might desensitize cytokinin perception as the cytokinin receptors expose their hormone-binding domain to the ER lumen (Schaller et al., 2015Schaller G.E. Bishopp A. Kieber J.J. The Yin-Yang of hormones: cytokinin and auxin interactions in plant development.Plant Cell. 2015; 27: 44-63Crossref PubMed Scopus (339) Google Scholar). Among the phosphoproteins with differential phosphorylation between the ahk2ahk3 mutant and WT in the kinetin dataset, we found an over-representation of the category “calcium signaling” (Figure 1A). Beside two calmodulin-like proteins (CML12: AT2G41100; CML25: AT1G24620) and two calcium-binding EF hand family proteins (AT5G28830, AT1G29020) of unknown physiological function, we identified one calmodulin-binding transcriptional regulator-like protein (AT4G16150) related to the Oryza sativa CaM-binding transcription factor (Choi et al., 2005Choi M.S. Kim M.C. Yoo J.H. Moon B.C. Koo S.C. Park B.O. Lee J.H. Koo Y.D. Han H.J. Lee S.Y. et al.Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).J. Biol. Chem. 2005; 280: 40820-40831Crossref PubMed Scopus (101) Google Scholar), one calcium-transporting ATPase of the E1–E2 type (ACA13), probably exporting calcium ions out of the cell (Iwano et al., 2014Iwano M. Igarashi M. Tarutani Y. Kaothien-Nakayama P. Nakayama H. Moriyama H. Yakabe R. Entani T. Shimosato-Asano H. Ueki M. et al.A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.Plant Cell. 2014; 26: 636-649Crossref PubMed Scopus (54) Google Scholar), and three calcium-dependent protein kinases (CPKs) (Figure 1C and 1D). Although the functional role of the phosphorylation/dephosphorylation of these proteins has yet to be elucidated, the AHK2/AHK3-dependent TCS appears to influence calcium signaling and homeostasis. We also identified 21 protein kinases among the AHK2/AHK3-dependent, differentially modified phosphoproteins in the kinetin data set (Figure 1D). One of these was KIN10, with an enhanced phosphorylation in the mutant at Thr6 and Tyr19, N-terminal to the kinase domain. KIN10 is one of the two main catalytic subunits of the Arabidopsis SNF1-related kinase 1 (SnRK1). Upon sensing energy stress, SnRK1 triggers both transcriptional and non-transcriptional changes that contribute to metabolic reprogramming and restoration of energy homeostasis (Baena-González and Sheen, 2008Baena-González E. Sheen J. Convergent energy and stress signalling.Trends Plant Sci. 2008; 13: 474-482Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Assuming that the phosphorylations trigger the activity of SnRK1, a link is established to how the AHK2/AHK3-dependent TCS could promote long-term effects in plant cell survival, differentiation, and development at the level of metabolic and energy homeostasis. Another three prominent kinases, which showed a reduced phosphorylation in the mutant compared with the WT in the kinetin dataset, are the three CPKs mentioned above, namely CPK4, CPK10, and CPK12 (Figure 1D and Supplemental Table 1). These CPKs function in abscisic acid, drought, and salinity signaling in a calcium-dependent manner (Boudsocq and Sheen, 2013Boudsocq M. Sheen J. CDPKs in immune and stress signalling.Trends Plant Sci. 2013; 18: 30-40Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Whether these modifications influence the activity of the CPKs is not yet known; however, one may hypothesize that the functional interaction of the AHK2/AHK3-dependent TCS signaling and abiotic stress responses (Zwack and Rashotte, 2015Zwack P.J. Rashotte A.M. Interactions between cytokinin signaling and abiotic stress responses.J. Exp. Bot. 2015; 66: 4863-4871Crossref PubMed Scopus (203) Google Scholar) might be the result of modification of CPKs. We also found the Arabidopsis MAP kinase 18 (ATMPK18) to have reduced phosphorylation in the mutant compared with the WT in the kinetin dataset (Figure 1D and Supplemental Table 1). The phosphorylations occur at Ser415 and Ser426, C-terminal to the kinase domain. Phosphorylation of S426 was also found in a phosphoproteomics study of DNA-repair mechanism (Roitinger et al., 2015Roitinger E. Hofer M. Köcher T. Pichler P. Novatchkova M. Yang J. Schlögelhofer P. Mechtler K. Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana.Mol. Cell. Proteomics. 2015; 14: 556-571Crossref PubMed Scopus (140) Google Scholar). Furthermore, ATMPK18 forms a signaling module with the dual-specificity MAPK phosphatase, PROPYZAMIDE HYPERSENSITIVE 1, which regulates cortical microtubule functions (Walia et al., 2009Walia A. Lee J.S. Wasteneys G. Ellis B. Arabidopsis mitogen-activated protein kinase MPK18 mediates cortical microtubule functions in plant cells.Plant J. 2009; 59: 565-575Crossref PubMed Scopus (72) Google Scholar). Consistently, a significant number of phosphoproteins with functions in “cell organization” were identified in the kinetin dataset, which is differentially modified in an AHK2/AHK3-dependent manner (Figure 1A). These proteins are mainly cytoskeletal components or are involved in processes such as cell division, cell organization, and vesicle trafficking (Figure 1E). With respect to cell division, we identified one cyclin-dependent protein kinase (CYCD7;1), one cyclin (CYCT1;1), two cell division cycle proteins (CDC48, AT3G53230), and a component of the SMC5/6 complex (SMC6A: AT5G07660) (Figure 1E). Regarding cell organization, three different myosin heavy chain-related proteins (AT3G42060, AT1G20400, AT1G64320), two ankyrin repeat-containing proteins (AT5G14230, AT2G04740), one putative actin-binding (AT5G48460) and one microtubuli-associated (MAP65-5) protein, as well as the kinesin-5 motor protein (ATK5) were found (Figure 1E). Finally, the katanin-like protein ERH3 and one syntaxin (SYP123) showed AHK2/AHK3-dependent differential modification. These results are in agreement with the function of the AHK2/AHK3-dependent TCS in the regulation of cell division and cell growth (Kieber and Schaller, 2014Kieber J.J. Schaller G.E. Cytokinins.Arabidopsis Book. 2014; 12: e0168Crossref Google Scholar), and may add a new level of complexity to the control of these cellular processes. The AHK2/AHK3-dependent changes in the Ser/Thr/Tyr phosphoproteome of Arabidopsis raise the question of how the AHK cytokinin receptors or their downstream TCS elements achieve this. From a thermodynamic point of view canonical HKs, HPs, and RRs are not able to phosphorylate Ser, Thr, or Tyr directly. Therefore, other mechanisms must exist that enable the AHK2/AHK3-dependent TCS elements to activate canonical Ser/Thr/Tyr kinases or phosphatases, which may then be responsible for the majority of protein modification found in our phosphoproteome data set. At least two possible and complementary mechanisms are conceivable, which have the physical interaction of the TCS elements with putative target kinases in common. In the first case an interaction with TCS elements may induce autophosphorylation followed by autoactivation of the target kinases. These kinases may cause an initial wave of phosphorylation. An example of this mechanism is the autophosphorylation and activation of the MAPKKKs Ssk2 by physical interaction with the canonical response regulator Ssk1 in the yeast Hog1 pathway (Posas and Saito, 1998Posas F. Saito H. Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator.EMBO J. 1998; 17: 1385-1394Crossref PubMed Scopus (250) Google Scholar). In the second case the association of TCS elements with kinases results in their activation without autophosphorylation. Such kinases are then responsible for the modification of secondary kinases (and phosphatases), which in turn phosphorylate downstream targets. An in planta example of this mechanism is the conformational regulation of MAPKKK-like constitutive triple response 1 (CTR1) by interaction with the ethylene receptors, members of the HK family (Wang et al., 2013Wang F. Cui X. Sun Y. Dong C.H. Ethylene signaling and regulation in plant growth and stress responses.Plant Cell Rep. 2013; 32: 1099-1109Crossref PubMed Scopus (111) Google Scholar). Both mechanisms could take place in the ER membrane, at the ER–cytosol interface, in the cytosol, or in the nucleus, as the TCS elements of the AHK2/AHK3 signaling pathway are found in all these compartments (Kieber and Schaller, 2014Kieber J.J. Schaller G.E. Cytokinins.Arabidopsis Book. 2014; 12: e0168Crossref Google Scholar). Interestingly, although the cytokinin receptor-like family of monocots consists for the most part of canonical HKs, O. sativa AtCRE1-like 4 (OsCRL4) is predicted to be a Ser/Thr kinase (Han et al., 2004Han Q.-M. Jiang H.-W. Qi X.-P. Yu J. Wu P. A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice.J. Zhejiang Univ. Sci. 2004; 5: 629-633Crossref PubMed Scopus (14) Google Scholar). This opens the possibility that at least in rice, OsCRL4 might be able to cause rapid changes in Ser/Thr phosphorylation in a direct manner. Our study revealed a previously unrecognized massive signaling transition from the AHK2/AHK3-dependent TCS phosphorelay to Ser/Thr/Tyr phosphorylation/dephosphorylation. In future, it will be worth exploring (1) how and at which molecular level within the TCS (HK, HP, or RR) and where in the cell this transition occurs, (2) what the consequences of the modification are for the functional properties of the identified proteins, and (3) how the entire phosphorelay–phosphorylation network is organized. This work was supported by a grant from the DFG to K.H. (SFB 1101, project B05) and by a fellowship from the Landesgraduiertenförderungsprogramm of Baden-Württemberg, Germany, to R.D.