Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of... Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2012, Article ID 581460, 9 pages doi:10.1155/2012/581460 Research Article Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas Jens Boesger, Volker Wagner, Wolfram Weisheit, and Maria Mittag Institute of General Botany and Plant Physiology, Friedrich Schiller University Jena, Am Planetarium 1, 07743 Jena, Germany Correspondence should be addressed to Maria Mittag, m.mittag@uni-jena.de Received 2 August 2012; Accepted 10 November 2012 Academic Editor: Jaroslav Dolezel ˇ Copyright © 2012 Jens Boesger et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. 1. Introduction components as well as proteins with homologues associated with human diseases (e.g., polycystic kidney disease, retinal Eukaryotic cilia or flagella are microtubule-based organelles degeneration, hydrocephalus, or changes in the left-right that are highly conserved in protein composition and symmetry of organs) collectively known as ciliopathies [5]. structural organization from protozoa to mammals. They But in many cases, Flagellar Associated Proteins (FAPs) still are structurally characterized by nine microtubular doublets have unknown function. surrounding two central microtubular singlets [1]. Substruc- Among the proteins in the flagellum, 21 protein kinases tures like dynein arms and radial spokes are associated with and 11 protein phosphatases were found pointing to regula- the axoneme and important for motility in the flagellum. tion by reversible protein phosphorylation in this organelle. Matrix proteins that are not tightly associated with the Phosphorylation events on specific amino acids residues flagellar membrane or the axoneme serve diverse functions in can affect protein function, its intracellular localization, its the flagellum and can be involved in intraflagellar transport activity, and its affinity to interaction partners (for review [2]. see [6]). But the identification of substrates for kinases Since many years, the green biflagellate alga Chlamy- in the phosphoregulatory pathway is still a challenge. In domonas reinhardtii, whose genome has been sequenced, is C. reinhardtii, several proteomes and phosphoproteomes of used as a model to study flagella structure, assembly, forma- subcellular compartments (reviewed in [7, 8]) were ana- tion, and motility [3]. C. reinhardtii uses flagella for motility lyzed including environmentally modulated photosynthetic in aqueous environments, for attaching to surfaces and for membranes [9], the eyespot [10], and the flagellum [11]. The flagellum phosphoproteome was first studied under cell-cell recognition during mating. A proteomic analysis of Chlamydomonas flagella revealed more than 600 proteins [4] physiological conditions without postincubation of isolated that include, for example, motor and signal transduction flagellar proteins with ATP to increase the phosphorylation 2 International Journal of Plant Genomics status. 126 in vivo phosphorylation sites were found belong- 2.2. Crude Extract Preparation and Immunodetection. Pro- ing to 32 different structural and motor proteins, several tein extracts were prepared as described previously [11]. The kinases, and proteins with protein interaction domains concentration of proteins was measured according to [22]. [11]. Furthermore, a dynamic phosphorylation pattern and Immunoblots were done with antibodies against phospho- clustering of phosphorylation sites were found in some Ser (Qiagen) and phosphoThr (Cell Signaling Technology) cases, indicating the specific control of proteins by reversible according to the manufacturer’s instructions. Polyclonal phosphorylation in the flagellum. In another study, flagellum antibodies against the C-terminal part of CK1 (amino acids phosphoproteins were examined during flagella shortening. 131–333 out of 333; ID JGI Vs3: 137286) were also used In this case, postincubation with ATP was undertaken. [23]. For this, the C-terminal part of CK1 was expressed Thereby, half of the identified phosphoproteins were only and purified from E. coli according to the Qiagen protocol. detected in shortening flagella [12]. Antibodies were raised by the “Pineda-Antikor ¨ per-Service,” The axoneme of Chlamydomonas flagella anchors mul- Berlin, Germany. Immunoblots were done as described [11] tiple inner arm dyneins and a network of kinases and using the polyclonal anti-CK1 antibody in a dilution of phosphatases that control motility by reversible protein 1 : 5,000. phosphorylation [13]. One of the involved flagellum kinases is Casein Kinase 1 (CK1) [14–16]. In pharmacological exper- 2.3. Densitometry Analysis. Quantifications were done with iments using a specific CK1 inhibitor (CKI-7), it was shown the Image Master 2D Elite (version 4.01) software from GE that CK1 regulates dynein activity and flagellum motility by Healthcare (formerly Amersham Pharmacia Biotech). phosphorylation of the Inner Dynein Arm I1 Intermediate Chain 138 (IC138) [14, 15]. Moreover, silencing of CK1 2.4. Measurement of Swimming Velocity of C. reinhardtii results in alterations of circadian phototaxis (shortening of Cells. Measurement of swimming velocity was done by using the period), defects in flagella formation, and in hatching a hemocytometer and a differential interference contrast of the daughter cells [17]. Interestingly, alterations in the microscope with a total magnification of 400 including a expression of several other key players of the clock machinery personal computer with a digital video recording system to of C. reinhardtii named Rhythm of Chloroplast (ROC) and measure displacement versus time. The swimming velocity a homologue of Constans (CrCO) have in parallel severe was determined manually by measuring the linear displace- effects on hatching, flagella formation, and/or movement, ment of cells on the scale of the micrometer. 10 samples were underlining that these processes are interconnected in C. measured to obtain the average velocity of a given sample. reinhardtii [17–19]. Regarding the multiple functions of CK1 in flagella 2.5. Cell Growth, CKI-7 Treatment, Isolation of Flagella, formation and motility along with its regulatory role in the Protein Digestion, and Enrichment of Phosphopeptides by circadian system in C. reinhardtii, we were interested in the Immobilized Metal Affinity Chromatography (IMAC). Cells identification of CK1 targets in flagella beside IC138. In a were growninaLD cycleand harvestedatthe endof comparative phosphoproteomic approach using wild-type −1 the night (LD24) at a cell density of 2-3 × 10 cells mL cells with and without CKI-7 treatment, we determined the by centrifugation (700×g, 5 min, 4 C).Cells were resus- targets of CK1 in the flagellum. In the CKI-7-treated cells, pended in one-half volume of minimal medium [21]and several phosphoproteins were missing or were identified with then the culture was kept under dim light conditions for a reduced number of phosphorylation sites, compared to 29 h representing subjective day (LL29), before cells were untreated wild-type cells. Also novel phosphopeptides or harvested (700×g, 15 min, 4 C). In some cases, the CK1 additional phosphorylation sites of known phosphopeptides inhibitor, CKI-7, (N-(2-Aminoethyl)-5-chloroisoquinoline- were identified in the CKI-7-treated cells, suggesting that 8-sulfonamide; Toronto Research Chemicals Inc.) [24], was CK1 is part of a signaling network in the flagellum. added to the culture to a final concentration of 50 μM following the shift to LL conditions. Isolation of the 2. Materials and Methods matrix membrane axoneme fraction (MMA) of flagella, tryptic digestion of MMA proteins, and enrichment of Standard molecular biology methods were done according to flagellum phosphopeptides by IMAC were done as previously [20]. described [11]. 2.1. Cell Culture. C. reinhardtii strain 137c (nit1 nit2)was 2.6. Peptide Identification by Nano-Liquid Chromatography- used with whom the flagellar proteome and phosphopro- Electrospray Ionization-Mass Spectrometry (nLC-ESI-MS) teome were analyzed [4, 11]. Cells were grown in TAP and Data Analysis. nLC-ESI-MS and data analysis were medium [21] under a 12 h light-12 h dark cycle (LD 12 : 12) carried out as described before [11]. Briefly, phosphopep- −2 −1 with a light intensity of 71 μEm sec (1 E = 1mol of tides were subjected to nLC-ESI-MS using an UltiMate photons) at 24 C. The beginning of the light period is 3000 nano-HPLC (Dionex Corporation) with a flow rate of −1 defined as time zero (LD0) and the beginning of the dark 300 nL min coupled online with a linear ion trap ESI-MS period is LD12. In some cases, cells were released after (Finnigian LTQ, Thermo Electron Corp.). The instrument growth in LD into constant conditions (LL) of dim light was run by the data-dependent neutral loss method, cycling −2 −1 (15 μEm sec ). between one full MS and MS/MS scans of the four most International Journal of Plant Genomics 3 abundant ions. After each cycle, these peptide masses were CK1 seems to lead to the activation of other kinases resulting excluded from the analysis for 10 sec. The detection of a in the phosphorylation of other proteins. neutral loss fragment (98, 49, or 32.66 Da) in the MS scans As mentioned before, flagellum kinases affect motility. triggered an MS scan of the neutral loss ion representing the We also studied if the inhibition by CKI-7 results in changes dephosphorylated peptide. in swimming velocity. To analyze the swimming behavior, we compared the swimming velocity of the C. reinhardtii strain Data analysis was done using the Proteome Discoverer 137c with cells that were cultivated with CKI-7 as described software (Version 1.0) from Thermo Electron Corp. includ- (see Section 2). Cells were spotted on a hemocytometer and ing the SEQUEST algorithm [25]. The software parameters the swimming velocity was measured using a differential were set to detect a modification of 79.96 Da in Ser, 2 3 interference contrast microscope including a personal com- Thr, or Tyr in MS and MS spectra. For the database puter with a video recording system (see Section 2). The searches with MS data, modifications of −18.00 Da on assay revealed that the swimming speed of CK1-inhibited Ser and Thr residues representing the neutral loss were cells is significantly lower (75.6 μm/s; ±4,1 SEM) compared additionally used. Further, detection of a modification of to untreated cells (122.2 μm/s; ±2.5 SEM) (Figure 1(d)). 16 Da on Met representing its oxidized form was enabled These data show that CK1-mediated phosphorylation events and carboxyamidomethylation of Cys residues was enabled in flagella influence motility and swimming speed of C. as a static modification. Peptide mass tolerance was set to 2 3 reinhardtii cells. 1.5 Da in MS mode. In MS and MS modes, fragment ion tolerance was set up to 1 Da. The parameters for all 3.2. The Flagellum Phosphoproteome of CKI-7-Treated Cells. database searches were set to achieve a false discovery rate The targets of CK1 in the flagellum are of high interest with (FDR) of not more than 1% for each individual analysis. regard to flagella formation as well as for clock control events. Data were searched against the flagellar proteome database They are largely unknown. An exception is IC138 that is [4](http://labs.umassmed.edu/chlamyfp/index.php). Addi- suggested as a direct target of CK1 based on experimental tionally, NCBI and the Joint Genome Institute C. reinhardtii data (summarized in [25]). databases (Version 2 and Version 3) were used for data In a next step, the direct and indirect targets of CK1 evaluation. were analyzed by a functional proteome approach. For that purpose, we compared the already existing phosphopro- 3. Results teome [11] with one investigated exactly under the same conditions with the single exception that CK1 is inhibited. 3.1. The Effects of the CK1 Inhibitor CKI-7 on the Phos- Since strong silencing of CK1 by RNAi results in defects phorylation Pattern of Flagellum Proteins and the Swimming in flagella formation, flagellum material cannot be obtained Velocity of C. reinhardtii. CK1 was found in the proteome in a significant amount from such strains [17]. Therefore, of the flagellum [4] and was also shown immunologically to inhibition of CK1 with CKI-7 was used. Cells were grown be enriched in flagella in wild-type strain SAG 73.72 [17]. under a light-dark cycle and the inhibitor was added for a For the comparative phosphoproteome analysis, flagella were 29 h period right at the moment when the cells were released isolated from strain 137c along with the dibucaine method to constant dim light. LL29 was also used as harvesting time [11]. We first examined the enrichment of CK1 in flagella point in the previous analysis [11]. of 137c using the applied conditions by immunodetection We avoided to add high amounts of ATP to isolated along with anti-CK1 antibodies (Figures 1(a) and 1(b)). Lev- flagella and to postincubate them at elevated temperatures els of CK1 were significantly enriched in the flagella fraction, to induce kinase activities in vitro, as done in another especially compared to cell bodies lacking flagella. Thus, the study [12]. We found that this treatment leads to severe procedure used for identification of the phosphoproteome phosphorylation events that include most likely phospho- maintains the enrichment of CK1 in flagella and is thus rylation steps that would not take place in vivo under suited to screen for its targets. physiological conditions See Supplemental Figures 1(a), In the next step, we examined to what degree the 1(b) in Supplementary Material available online at doi: CK1-specific inhibitor, CKI-7 [24], which was already used 1155/2012/581460. for studying CK1 in C. reinhardtii [15], influences the The further analysis of the phosphoproteome in CKI-7- phosphorylation pattern of flagellum proteins. Therefore, we treated cells was carried out with the same procedure and grew cells with and without CKI-7 treatment, respectively, criteria as applied before for the flagellum phosphoproteome and compared the flagellum phosphoproteins from both [11]. Three independent isolations of flagella of CKI-7- aliquots by immunodetection with antiphosphoSer anti- inhibited cells were carried out and subjected to phospho- bodies (Figure 1(c)). As expected, several phosphorylated peptide purification along with liquid chromatography mass protein bands were reduced to a significant extent or spectrometry (for details, see [11]). Previously identified absent in the CKI-7-inhibited cells (Figure 1(c), labeled with phosphopeptides or phosphorylation sites within a phospho- “−”). At the same time, some phosphoprotein bands were peptide ( listed in Table S1 in [11]) that had not been detected stronger (Figure 1(c), labeled with “+”). These data show in any of the three analyses were considered to be either direct that inhibition of CK1 has a dual effect. On the one hand, or indirect targets of CK1. The phosphoproteins to which the phosphorylation of CK1 targets drops strongly down or these phosphopeptides belong are listed in Table 1.Novel is fully stopped by its inhibition; on the other hand, inactive phosphopeptides belonging to novel phosphoproteins that 4 International Journal of Plant Genomics CK1 CE CB FL CE CB FL (a) (b) CKI-7 − + − 60 −CKI-7 +CKI-7 (c) (d) Figure 1: Enrichment of CK1 in flagella and the influence of CK1 inhibition on the phosphorylation status of flagellum proteins and swimming velocity of C. reinhardtii cells. (a) Cells were grown in TAP in a 12 h light-12 h dark cycle and then released to dim light (LL) according to Section 2. Cells were harvested at LL29 and flagella were isolated and a whole cell crude extract (CE), a flagellar extract (FL), and an extract from cell bodies lacking flagella (CB) were prepared. 25 μg proteins per fraction were separated by SDS-PAGE and analyzed by immunoblotting with anti-CK1 antibodies according to Section 2. (b) For quantifications, the amount of CK1 detected in the whole cell crude extract was set to 100%. Quantifications were done with three biological replicates using the ImageMaster 2D Elite Vs.4.01 software (GE Healthcare). (c) Changes in the phosphorylation pattern of flagellum proteins in cells treated with and without CKI-7. Cells were grown as described above (a) in the presence or absence of CKI-7 and harvested at LL29 before isolation of flagella. Proteins from the MMA fraction of the flagellum (25 μg each lane) were separated by 9% SDS-PAGE along with a molecular mass standard and immunoblotted with specific antibodies against phosphoSer according to Section 2. Changes in the phosphorylation status of proteins after CKI-7 treatment are indicated by “+” and “−” signs, respectively. (d) Swimming velocity of 137c cells in the absence (−CKI-7) or presence of CK1 inhibitor (+CKI-7). Cells were grown at 23 C in a LD cycle. Measurements of swimming velocity were done with a hemocytometer and a differential interference contrast microscope with a total magnification of 400 including a personal computer with a video recording system to measure displacement versus time (n = 10). Error bars represent the SEM. had not been identified in the former analysis and additional different combinatory phosphorylation patterns (data not phosphopeptides or phosphorylation sites of already identi- shown). fied phosphoproteins are listed in Table 2. Details about all In the CKI-7-treated cells, phosphopeptides from 14 newly identified peptides and phosphorylation sites can be phosphoproteins were missing (Table 1). Four additional found in Supplemental Table S1. In three cases, (TEKTIN, phosphoproteins were identified again but with a reduced FAP18, and FAP262), all previous identified phosphorylation number of phosphorylation sites. These are labeled by sites were detected again, but in some phosphopeptides with indices along with the missing sites in Table 1.Among (kDa) Swimming velocity (µm/s) Relative abundance (%) International Journal of Plant Genomics 5 KLP1 PF6 C1 C2 IFT43 H RSP11 RSP17 IC138 DC2 DC1 (a) RSP11 0 204 RIIa 30 31 32 33 34 35 36 37 38 39 40 41 RQ P Tp DL I A F SA K (b) GSK3 Ser/Thr kin 231 232 233 234 235 236 237 238 239 240 241 242 243 244 I I CS R L KE G P N SYp L KE G P NI Sp Y I CS R (c) CK1 Kinase GSK3 (active) (inactive) (inactive) (d) CKI-7 GSK3 CK1 Kinase (active) (active) (inactive) (e) Figure 2: Analysis of CK1 targets in the flagellum. (a) Diagram of flagellum phosphoproteins in wild-type and CK1-inhibited cells. A cross-section of a flagellum from C. reinhardtii (left panel) and a more detailed view (red rectangle) are shown according to [11]. Structural phosphoproteins in CKI-7-inhibited cells, and such with a reduced number of phosphorylation sites are indicated in yellow color with a red frame. Novel phosphopeptides of structural proteins or additional phosphorylation sites of known phosphopeptides from structural phosphoproteins that were identified in the CKI-7-inhibited proteome are indicated by yellow color with a blue frame. Structural proteins with previously identified phosphopeptides, whose phosphorylation sites were detected again, are indicated in yellow without frame. Abbreviations are: C1 central pair projection (C1P), C2 central pair projection (C2P), PF6 protein (PF6), Hydin (H), Radial Spoke Protein17 (RSP17); Outer Dynein Arm Docking Complex (DC); Inner Dynein Arm Intermedite Chain138 (IC138), Tectin (T) as well as an Intraflagellar Transport Protein43 (IFT43). (b) and (c) Positions of identified phosphopeptides in the predicted domains of RSP11 (b) and GSK3 (c). Identified phosphopeptides are indicted by black boxes. The amino acid positions are mentioned. “p” indicates in vivo phosphorylation sites. RIIa, regulatory subunit of cAMP-Dependent Protein Kinase A; Ser/Thr Kin, Ser/Thr protein kinase catalytic domain. (d) and (e) Hypothetical model of GSK3 de-/activation via reversible phosphorylation triggered by CK1. (d) Regulatory signaling involves an additional kinase. The noninhibited active form of CK1 inactivates another still unknown kinase by phosphorylation. This kinase is needed in its active nonphosphorylated form for activating GSK3. (e) If CK1 is inhibited by CKI-7, the unknown kinase is not phosphorylated and thus active. This active kinase phosphorylates in turn GSK3, which is then activated. 6 International Journal of Plant Genomics Table 1: Phosphoproteins identified in 137c [11] whose phospho- Table 2: Additional phosphopeptides/phosphorylation sites in peptides or phosphorylation sites are missing in CKI-7-treated cells. CKI-7-treated cells of either novel phosphoproteins or phosphopro- teins that were already identified in 137c [11]. Flagellar central pair-associated protein; PF6 Phosphoproteins only present in CKI-7-treated 137c cells Hydin-like protein; HYD3 Glycogen synthase kinase 3; GSK3 Inner dynein arm I1 intermediate chain; IC138 Kinesin-like protein; Kinesin motor domain, KIF9-like subgroup Intraflagellar transport protein IFT43 a,b Phosphoglucomutase Outer dynein arm docking complex subunit 1 ;ODA-DC1, ODA3 Radial spoke protein 11; RSP11; RIIa domain Radial spoke protein 17; RSP17 S-Adenosylmethionine synthetase FAP59 ; RecF/RecN/SMC N-terminal domain FAP139 ; TIGR02680 domain a,d,e FAP116 ; microtubule-binding protein MIP-T3 domain FAP21 a,f FAP190 ; sterile alpha motif FAP56 FAP228; callose synthase-like protein; 1,3-beta-glucan synthase FAP75 component FAP98 FAP230; ankyrin repeats; ion transport protein domain FAP129 FAP254; putative ankyrin-like protein FAP165 FAP288; EF hand FAP236 a,g FAP1 FAP241 FAP93 FAP243 (Vs3 FAP183) FAP147 Phosphoproteins found in CKI-7-treated cells with additional FAP184 phosphopeptide(s) in comparison to 137c FAP263 Outer dynein arm docking complex protein 2; ODA-DC2 The function of depicted proteins is given as determined by NCBI BLASTp, a,b FAP33 ; ankyrin repeats along with their conserved domains. FAP154 Not all previously identified peptides (listed in Table S1 in [11]) are present in the CKI-7-treated cells. FAP217 Variants of peptide TISGADTPEEVLAYWEGLK with the phosphorylation Phosphoproteins found in CKI-7-treated cells with the same sites Thr-345, Ser-347, and Thr-351 as well as variants of peptide ILGYTGS- peptide [11] but with additional phosphorylation site(s) DVEEEEPESEEETEEEANKDDGVVDR with the phosphorylation sites Tyr- 697 and Ser-709 are missing. FAP39 ; plasma membrane calcium transporting ATPase Predicted functional domains are present only in the Vs3 model. d MAK7 ; mitogen activated protein kinase 7 Vs2 model differs significantly from Vs3 model. The phosphorylation site Ser-255 in peptide SASPGGEDPLNKSGSAAPK The function of depicted proteins is given as determined by NCBI BLASTp, is missing. along with their conserved domains. Variants of peptide STSSIGGGYSEPVGSDGEGSDAASAKPR with phos- Vs2 model differs significantly from Vs3 model. phorylation sites on Ser-370, Ser-375 and Ser-379 are missing. Predicted functional domains are present only in the Vs3 model. The phosphorylation site Ser-55 in peptide SRGSFQEGQAMVR is missing. Protein Kinase A (PKA) and bears a phosphorylation site these 18 phosphoproteins, six known structural proteins are (Figure 2(b)). Two other kinases were also found in this present including IC138 that was suggested to be a direct category. One of them is Glycogen Synthase Kinase 3 (GSK3). target of CK1 [26]. All missing structural phosphoproteins as The level of active GSK3 is postulated to be regulated via well as those with a reduced number of phosphorylation sites phosphorylation of a conserved Tyr correlating with flagellar are indicated in yellow color with a red frame in Figure 2(a). length [27]. Exactly this Tyr that is situated in the Ser/Thr Moreover, seven FAPs with conserved domains are affected kinase domain of GSK3 is phosphorylated as well as a in the CKI-7-treated phosphoproteome as well as five FAPs Ser in its surroundings (Supplemental Table 1; Figure 2(c)). without any conserved domains. Notable GSK3 is also clock relevant, for example, in Also novel phosphopeptides or additional phosphory- Drosophila [28]. Moreover, a Mitogen Activated Kinase, lation sites of known phosphopeptides were identified in MAK7, was found with additional phosphorylation sites. the proteome of CKI-7-treated cells, suggesting that CK1 is part of a signaling network in the flagellum. They belong 4. Discussion to either 15 new phosphoproteins or six already known phosphoproteins (Table 2, Supplemental Table 1). Among The identification of targets of CK1 in the flagellum will them, some structural components are present, indicated by help understanding flagella formation as well as clock yellow color with a blue frame in Figure 2(a).Thereby,Radial control events related to flagella [17–19]. The fact that Spoke Protein 11 (RSP11) is of special interest. It has an RIIa several phosphorylated flagellar protein bands disappear in domain, which is a regulatory subunit of cAMP Dependent CKI-7-treated cells suggests that CK1 has multiple targets International Journal of Plant Genomics 7 in the flagellum. Among the 32 phosphoproteins of the was reduced to a similar degree in comparison to the flagellum, 14 were missing in the flagellum phosphopro- mutant strains that are lacking IC138, suggesting that the teome when the CKI-7 inhibitor was used or represented generation of flagellum motility is regulated by a CK1- with a reduced number of phosphorylation sites (four mediated phosphorylation of IC138 as suggested before [14, cases, Table 1). Missing phosphorylation sites cannot be 15]. automatically considered as direct targets of CK1. It could Another structural phosphoprotein previously identified be that the phosphorylation of an amino acid residue by with two phosphopeptides and variable phosphorylation CK1 represents a trigger that then allows a consequent sites is ODA-DC1. The outer dynein arm docking complex phosphorylation of another amino acid residue in the (ODA-DC), which is composed of three proteins, designated surroundings by another kinase. An example for consequent DC1, DC2, and DC3, is associated with microtubules phosphorylation steps of different kinases is mentioned and targets the outer dynein arms to its binding site on below and involves PKA, GSK3, and CK1. Also, CK1 may the flagellum axoneme [33]. In both previously identified activate or deactivate another kinase by reversible phos- phosphopeptides certain phosphorylation sites are missing phorylation. In the current study, the previously identified in CKI-7-inhibited cells (Table 1; indices a, b) pointing out kinases along with their phosphorylation sites were found that they are CK1 targets. ODA-DC2 had been also identified again [11]. Only in case of FAP262 that bears a Ser/Thr kinase in the previous study [11] with one phosphopeptide and domain, a different combinatory phosphorylation pattern variable phosphorylation sites, which were all found again was observed, which might be relevant. But it could also in the current study. But now a novel phosphopeptide with be that some of the missing phosphoproteins in the FAP phosphorylation on Ser-278 was present in CKI-7 cells, category whose functions are not known may have kinase underlining that CK1 seems to be indirectly involved in activity. Networks that consist of interconnected kinases regulating further kinases. along with protein phosphatases are not unusual in signaling. Radial spokes represent a major structural feature of 9+2 In line with this, we found also 21 new phosphoproteins axonemes and they are essential for flagellum beating. Each along with novel phosphopeptides or phosphorylation sites, radial spoke consists of a thin stalk, which is attached to including three kinase-related proteins. The presence of new the A-tubule of the axonemal doublet microtubules and a phosphorylation sites in flagella of CKI-7-inhibited cells head projecting toward the central apparatus [34]. The radial was already predictable from the appearance of novel flag- spoke of C. reinhardtii is composed of at least 23 proteins, ellar phosphoprotein bands detected with anti-phosphoSer and not all of them have been characterized at the molecular antibodies (Figure 1(c)). In this category, we identified level [35]. RSP17, which is located in the spoke stalk, two phosphoproteins involved in carbohydrate and amino was identified in the flagellum phosphoproteome analysis acid metabolism, respectively (Table 2). One of them, with two different phosphopeptides [11]. The absence of phosphoglucomutase, catalyzes the bidirectional conversion both phosphopeptides in CKI-7-treated cells suggests that of glucose-1-phosphate to glucose-6-phosphate. Glucose-1- RSP17 is at the same time a direct and/or indirect target phosphate can be transferred into glycolysis by this way. The of CK1. Functional domains in radial spoke proteins reveal flagellum contains all enzymes of the late glycolytic pathway; their role in mediating signaling pathways. For instance, they are able to generate ATP for direct use in the flagellum RSP11 consists of a regulatory subunit (RIIa) of PKA [35]. [4]. In mammals, the activity of phosphoglucomutase is However, RSP11 lacks the cAMP-binding domains of the RII regulated by phosphorylation [29]. The other metabolically regulatory subunit. We could identify RSP11 in the CKI- relevant enzyme in this category is S-adenosylmethionine 7-treated cells as a new phosphoprotein with one in vivo synthetase, a key enzyme of methionine metabolism. In rat phosphorylation site at Thr-35, which is located directly liver, the activity of the S-adenosylmethionine synthetase is in the RIIa domain (Figure 2(b)). The interaction between regulated by Protein Kinase C [30]. RII and A-kinase anchoring protein motifs (AKAP) can be One of the direct targets of CK1 was suggested to be regulated by phosphorylation of RII [36, 37]. A pharma- IC138, the Inner Dynein Arm I1 Intermediate Chain 138. cological analysis using an inhibitor and the RII regulatory It was shown that phosphorylation of IC138 correlates with subunits had detected an axonemal PKA activity [38]. But the inhibition of dynein activity and that PKA beside CK1 as PKA could not be found in the flagellar proteome in contrast well as the Protein Phosphatases PP2A and PP1 are involved to CK1, PP1, and PP2A [4]. Thus, it was hypothesized that there (summarized in [26]). IC138 was identified in CK1 C. reinhardtii could express a PKA with an unconventional active cells with one phosphopeptide that is situated at its structure [39]. The identified phosphorylation site within the N-terminus including variable phosphorylation sites [11]. RII subunit of RSP11 may be relevant in this context. None of these phosphorylation sites were detected after An additional flagellum kinase is GSK3 whose enzymatic CKI-7 treatment, underlining that IC138 is a direct and/or activity is inhibited by lithium causing flagellar elongation indirect target of CK1. A pharmacological analysis using [27]. It is known that GSK3 has a Tyr-phosphorylated, active CKI-7 revealed the impact of CK1 on IC138 phosphorylation form and is enriched in flagella. GSK3 is associated with [14]. This mechanism authorizes CK1 to regulate dynein the axoneme in a phosphorylation-dependent manner. The activity and control flagellum motility. Also an analysis of levelofactiveGSK3correlateswithflagellarlength[27]. We mutants lacking the IC138 subcomplex revealed strains that could identify the Tyr-240-phosphorylated GSK3 as well as swim forward with reduced swimming velocities [31, 32]. a Ser-239-phosphorylated alternative in the CKI-7-treated Interestingly, the swimming speed of the CKI-7-treated cells cells (Figure 2(c)), suggesting that inhibition of CK1 causes 8 International Journal of Plant Genomics activation of GSK3. Both in vivo phosphorylation sites are [3] S. S. Merchant, S. E. Prochnik, O. Vallon et al., “The Chlamydomonas genome reveals the evolution of key animal located in the catalytic kinase domain, which could play and plant functions,” Science, vol. 318, no. 5848, pp. 245–251, important roles in the regulation of the activity of GSK3 within signaling pathways. Notably, interplay between CK1 [4] G. J. Pazour, N. Agrin, J. Leszyk, and G. B. Witman, “Pro- and GSK3 is known for Hedgehog signaling pathways [40]. teomic analysis of a eukaryotic cilium,” The Journal of Cell Thereby, the Cubitus Interruptus (Ci-155) transcriptional Biology, vol. 170, no. 1, pp. 103–113, 2005. activator is involved. Ci-155 proteolysis depends on phos- [5] W. F. Marshall, “The cell biological basis of ciliary disease,” The phorylation at three sites of PKA. Then, these phosphoSer Journal of Cell Biology, vol. 180, no. 1, pp. 17–21, 2008. prime further phosphorylation at GSK3 and CK1 sites. This [6] J. Reinders and A. Sickmann, “Modificomics: posttranslational principle is a good example for consecutive phosphorylation modifications beyond protein phosphorylation and glycosyla- steps of different kinases as mentioned before. tion,” Biomolecular Engineering, vol. 24, no. 2, pp. 169–177, Several studies have shown that reversible phosphoryla- tion of Tyr causes increases and decreases in GSK3 kinase [7] N. Rolland, A. Atteia, P. Decottignies et al., “Chlamydomonas activity, respectively [41, 42]. For the interplay of CK1 proteomics,” Current Opinion in Microbiology, vol. 12, no. 3, and GSK3 in the C. reinhardtii flagella, one can imagine pp. 285–291, 2009. a regulatory mechanism, involving, for example, an addi- [8] V. Wagner, J. Boesger, and M. Mittag, “Sub-proteome analysis tional kinase. In a hypothetical model (Figure 2(d)), the in the green flagellate alga Chlamydomonas reinhardtii,” Jour- noninhibited, active CK1 inactivates another kinase by nal of Basic Microbiology, vol. 49, no. 1, pp. 32–41, 2009. phosphorylation, which is responsible for the activation [9] A. V. Vener, “Environmentally modulated phosphorylation of GSK3 by Tyr-phosphorylation. If CKI-7 inhibits CK1 and dynamics of proteins in photosynthetic membranes,” Bio- (Figure 2(e)), the additional kinase can stay active, because chimica et Biophysica Acta, vol. 1767, no. 6, pp. 449–457, 2007. it is not phosphorylated by CK1 and consequently GSK3 gets [10] V. Wagner, K. Ullmann, A. Mollwo, M. Kaminski, M. Mittag, and G. Kreimer, “The phosphoproteome of a Chlamydomonas converted to the phosphorylated active form. reinhardtii eyespot fraction includes key proteins of the light GSK3 plays also an important role in the regulation signaling pathway,” Plant Physiology, vol. 146, no. 2, pp. 772– of circadian systems. Shaggy (SGG), for example, the 788, 2008. Drosophila homologue of GSK3, is a central player in deter- [11] J. Boesger, V. Wagner, W. Weisheit, and M. Mittag, “Analysis of mining period length in flies by phosphorylation of clock flagellar phosphoproteins from Chlamydomonas reinhardtii,” components [43]. In mammals, GSK3 is proposed to phos- Eukaryotic Cell, vol. 8, no. 7, pp. 922–932, 2009. phorylate Clock (CLK), which is a core transcription factor [12] J. Pan, B. Naumann-Busch, L. Wang et al., “Protein phospho- that is essential for circadian behavior. Phosphorylation of rylation is a key event of flagellar disassembly revealed by anal- CLK controls its activity and degradation [44]. Especially ysis of flagellar phosphoproteins during flagellar shortening in kinases and phosphatases, which are relevant in regulating Chlamydomonas,” Journal of Proteome Research, vol. 10, no. 8, circadian clocks in other organisms, are well conserved in pp. 3830–3839, 2011. Chlamydomonas [45]. Interestingly, many output rhythms [13] M. E. Porter and W. S. Sale, “The 9 + 2 axoneme anchors that can be measured like phototaxis, chemotaxis, and multiple inner arm dyneins and a network of kinases and stickiness to glass and mating during the cell cycle involve phosphatases that control motility,” The Journal of Cell Biology, flagella. It is remarkable that kinases like CK1 or GSK3 as vol. 151, no. 5, pp. F37–F42, 2000. well as phosphatases like PP1 and PP2A are physically located [14] P. Yang and W. S. Sale, “Casein kinase I is anchored on in the axoneme [4, 26]. This underlines the important axonemal doublet microtubules and regulates flagellar dynein regulatory function of these components in the flagellum phosphorylation and activity,” The Journal of Biological Chem- istry, vol. 275, no. 25, pp. 18905–18912, 2000. regarding circadian rhythms. [15] A. Gokhale, M. Wirschell, and W. S. Sale, “Regulation of dynein-driven microtubule sliding by the axonemal protein Acknowledgments kinase CK1 in Chlamydomonas flagella,” The Journal of Cell Biology, vol. 186, no. 6, pp. 817–824, 2009. The authors thank the Joint Genome Institute (JGI) in the [16] M. Wirschell, R. Yamamoto, L. Alford, A. Gokhale, A. Gaillard, USA and the Kazusa Institute in Japan for the free delivery of and W. S. Sale, “Regulation of ciliary motility: conserved EST and genome sequences. This study was supported by the protein kinases and phosphatases are targeted and anchored in Deutsche Forschungsgemeinschaft (Grants Mi 373 to MM) the ciliary axoneme,” Archives of Biochemistry and Biophysics, and the BMBF (Project GoFORSYS, Grant no. 0315260A, vol. 510, no. 2, pp. 93–100, 2011. work package to MM). [17] M. Schmidt, G. Geßner, M. Luff et al., “Proteomic analysis of the eyespot of Chlamydomonas reinhardtii provides novel insights into its components and tactic movements,” The Plant References Cell, vol. 18, no. 8, pp. 1908–1930, 2006. [18] T. Matsuo, K. Okamoto, K. Onai, Y. Niwa, K. Shimogawara, [1] G. J. Pazour and G. B. Witman, “The vertebrate primary and M. Ishiura, “A systematic forward genetic analysis iden- cilium is a sensory organelle,” Current Opinion in Cell Biology, tified components of the Chlamydomonas circadian system,” vol. 15, no. 1, pp. 105–110, 2003. Genes and Development, vol. 22, no. 7, pp. 918–930, 2008. [2] J. L. Rosenbaum and G. B. Witman, “Intraflagellar transport,” [19] G. Serrano, R. Herrera-Palau, J. M. Romero, A. Serrano, G. Nature Reviews Molecular Cell Biology, vol. 3, no. 11, pp. 813– Coupland, and F. Valverde, “Chlamydomonas CONSTANS 825, 2002. International Journal of Plant Genomics 9 and the evolution of plant photoperiodic signaling,” Current [36] G. Keryer,Z.Luo,J.C.Cavadore, J. Erlichman, andM. Biology, vol. 19, no. 5, pp. 359–368, 2009. Bornens, “Phosphorylation of the regulatory subunit of type [20] J. Sambrook andD.W.Russel, Molecular Cloning: A Laboratory IIβ cAMP-dependent protein kinase by cyclin B/p34(cdc2) Manual, Cold Spring Harbor Laboratory Press, Cold Spring kinase impairs its binding to microtubule-associated protein Harbor, NY, USA, 2001. 2,” Proceedings of the National Academy of Sciences of the United [21] E. H. Harris, The Chlamydomonas Sourcebook,Academic States of America, vol. 90, no. 12, pp. 5418–5422, 1993. Press, San Diego, Calif, USA, 1989. [37] S. Manni, J. H. Mauban, C. W. Ward, and M. Bond, “Phospho- [22] V. Neuhoff, K. Philipp, H. G. Zimmer, and S. Mesecke, “A rylation of the cAMP-dependent protein kinase (PKA) reg- simple, versatile, sensitive and volume-independent method ulatory subunit modulates PKA-AKAP interaction, substrate for quantitative protein determination which is independent phosphorylation, and calcium signaling in cardiac cells,” The of other external influences,” Hoppe-Seyler’s Zeitschrift fur ¨ Journal of Biological Chemistry, vol. 283, no. 35, pp. 24145– Physiologische Chemie, vol. 360, no. 11, pp. 1657–1670, 1979. 24154, 2008. [23] T. Schulze, S. Schreiber, D. Iliev et al., “The heme-binding [38] A. R. Gaillard, L. A. Fox, J. M. Rhea, B. Craige, and W. S. Sale, protein SOUL3 of Chlamydomonas reinhardtii influences size “Disruption of the A-kinase anchoring domain in flagellar and position of the eyespot,” Molecular Plant. In press. radial spoke protein 3 results in unregulated axonemal cAMP- [24] F. Preuss, J. Y. Fan, M. Kalive et al., “Drosophila doubletime dependent protein kinase activity and abnormal flagellar mutations which either shorten or lengthen the period of motility,” Molecular Biology of the Cell, vol. 17, no. 6, pp. 2626– circadian rhythms decrease the protein kinase activity of 2635, 2006. casein kinase I,” Molecular and Cellular Biology, vol. 24, no. [39] E. H. Harris, The Chlamydomonas Sourcebook, vol. 3, Aca- 2, pp. 886–898, 2004. demic Press, San Diego, Calif, USA, 2009. [25] A. J. Link, J. Eng, D. M. Schieltz et al., “Direct analysis of [40] M. A. Price and D. Kalderon, “Proteolysis of the Hedgehog sig- protein complexes using mass spectrometry,” Nature Biotech- naling effector Cubitus interruptus requires phosphorylation nology, vol. 17, no. 7, pp. 676–682, 1999. by Glycogen Synthase Kinase 3 and Casein Kinase 1,” Cell, vol. [26] M. Wirschell, T. Hendrickson, and W. S. Sale, “Keeping an eye 108, no. 6, pp. 823–835, 2002. on I1:I1 dynein as a model for flagellar dynein assembly and regulation,” Cell Motility and the Cytoskeleton,vol. 64, no.8, [41] L. Kim, J. Liu, and A. R. Kimmel, “The novel tyrosine kinase pp. 569–579, 2007. ZAK1 activates GSK3 to direct cell fate specification,” Cell, vol. 99, no. 4, pp. 399–408, 1999. [27] N. F. Wilson and P. A. Lefebvre, “Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas [42] H. Murai, M. Okazaki, and A. Kikuchi, “Tyrosine dephos- reinhardtii,” Eukaryotic Cell, vol. 3, no. 5, pp. 1307–1319, 2004. phorylation of glycogen synthase kinase-3 is involved in its [28] E. Harms, M. W. Young, and L. Saez, “CK1 and GSK3 in extracellular signal-dependent inactivation,” FEBS Letters, vol. the Drosophila and mammalian circadian clock,” Novartis 392, no. 2, pp. 153–160, 1996. Foundation Symposium, vol. 253, pp. 267–277, 2003. [43] S. Panda, J. B. Hogenesch, and S. A. Kay, “Circadian rhythms [29] A. Gururaj, C. J. Barnes, R. K. Vadlamudi, and R. Kumar, from flies to human,” Nature, vol. 417, no. 6886, pp. 329–335, “Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase,” Oncogene, vol. 23, no. 49, pp. [44] M. L. Spengler, K. K. Kuropatwinski, M. Schumer, and M. 8118–8127, 2004. P. Antoch, “A serine cluster mediates BMAL1-dependent [30] M. A. Pajares, C. Duran, F. Corrales, and J. M. Mato, “Protein CLOCK phosphorylation and degradation,” Cell Cycle, vol. 8, kinase C phosphorylation of rat liver S-adenosylmethionine no. 24, pp. 4138–4146, 2009. synthetase: dissociation and production of an active mono- [45] M. Mittag, S. Kiaulehn, and C. H. Johnson, “The circadian mer,” Biochemical Journal, vol. 303, no. 3, pp. 949–955, 1994. clock in Chlamydomonas reinhardtii. What is it for? What is [31] T. W. Hendrickson, C. A. Perrone, P. Griffin et al., “IC138 it similar to?” Plant Physiology, vol. 137, no. 2, pp. 399–409, is a WD-repeat dynein intermediate chain required for light chain assembly and regulation of flagellar bending,” Molecular Biology of the Cell, vol. 15, no. 12, pp. 5431–5442, 2004. [32] K. E. VanderWaal, R. Yamamoto, K. Wakabayashi et al., “bop5 mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric wave- forms,” Molecular Biology of the Cell, vol. 22, no. 16, pp. 2862– 2874, 2011. [33] S. Takada, C. G. Wilkerson, K. I. Wakabayashi, R. Kamiya, and G. B. Witman, “The outer dynein arm-docking complex: com- position and characterization of a subunit (Oda1) necessary for outer arm assembly,” Molecular Biology of the Cell, vol. 13, no. 3, pp. 1015–1029, 2002. [34] A. M. Curry and J. L. Rosenbaum, “Flagellar radial spoke: a model molecular genetic system for studying organelle assembly,” Cell Motility and the Cytoskeleton,vol. 24, no.4,pp. 224–232, 1993. [35] P. Yang, D. R. Diener, C. Yang et al., “Radial spoke proteins of Chlamydomonas flagella,” JournalofCellScience, vol. 119, part 6, pp. 1165–1174, 2006. International Journal of Peptides Advances in International Journal of BioMed Stem Cells Virolog y Research International International Genomics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Nucleic Acids International Journal of Zoology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific Journal of Signal Transduction World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Advances in Genetics Anatomy Biochemistry Research International Research International Microbiology Research International Bioinformatics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Enzyme Journal of International Journal of Molecular Biology Archaea Research Evolutionary Biology International Marine Biology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Plant Genomics Hindawi Publishing Corporation

Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

Loading next page...
 
/lp/hindawi-publishing-corporation/application-of-phosphoproteomics-to-find-targets-of-casein-kinase-1-in-vq70hiKvBq
Publisher
Hindawi Publishing Corporation
Copyright
Copyright © 2012 Jens Boesger et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN
1687-5370
DOI
10.1155/2012/581460
Publisher site
See Article on Publisher Site

Abstract

Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2012, Article ID 581460, 9 pages doi:10.1155/2012/581460 Research Article Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas Jens Boesger, Volker Wagner, Wolfram Weisheit, and Maria Mittag Institute of General Botany and Plant Physiology, Friedrich Schiller University Jena, Am Planetarium 1, 07743 Jena, Germany Correspondence should be addressed to Maria Mittag, m.mittag@uni-jena.de Received 2 August 2012; Accepted 10 November 2012 Academic Editor: Jaroslav Dolezel ˇ Copyright © 2012 Jens Boesger et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. 1. Introduction components as well as proteins with homologues associated with human diseases (e.g., polycystic kidney disease, retinal Eukaryotic cilia or flagella are microtubule-based organelles degeneration, hydrocephalus, or changes in the left-right that are highly conserved in protein composition and symmetry of organs) collectively known as ciliopathies [5]. structural organization from protozoa to mammals. They But in many cases, Flagellar Associated Proteins (FAPs) still are structurally characterized by nine microtubular doublets have unknown function. surrounding two central microtubular singlets [1]. Substruc- Among the proteins in the flagellum, 21 protein kinases tures like dynein arms and radial spokes are associated with and 11 protein phosphatases were found pointing to regula- the axoneme and important for motility in the flagellum. tion by reversible protein phosphorylation in this organelle. Matrix proteins that are not tightly associated with the Phosphorylation events on specific amino acids residues flagellar membrane or the axoneme serve diverse functions in can affect protein function, its intracellular localization, its the flagellum and can be involved in intraflagellar transport activity, and its affinity to interaction partners (for review [2]. see [6]). But the identification of substrates for kinases Since many years, the green biflagellate alga Chlamy- in the phosphoregulatory pathway is still a challenge. In domonas reinhardtii, whose genome has been sequenced, is C. reinhardtii, several proteomes and phosphoproteomes of used as a model to study flagella structure, assembly, forma- subcellular compartments (reviewed in [7, 8]) were ana- tion, and motility [3]. C. reinhardtii uses flagella for motility lyzed including environmentally modulated photosynthetic in aqueous environments, for attaching to surfaces and for membranes [9], the eyespot [10], and the flagellum [11]. The flagellum phosphoproteome was first studied under cell-cell recognition during mating. A proteomic analysis of Chlamydomonas flagella revealed more than 600 proteins [4] physiological conditions without postincubation of isolated that include, for example, motor and signal transduction flagellar proteins with ATP to increase the phosphorylation 2 International Journal of Plant Genomics status. 126 in vivo phosphorylation sites were found belong- 2.2. Crude Extract Preparation and Immunodetection. Pro- ing to 32 different structural and motor proteins, several tein extracts were prepared as described previously [11]. The kinases, and proteins with protein interaction domains concentration of proteins was measured according to [22]. [11]. Furthermore, a dynamic phosphorylation pattern and Immunoblots were done with antibodies against phospho- clustering of phosphorylation sites were found in some Ser (Qiagen) and phosphoThr (Cell Signaling Technology) cases, indicating the specific control of proteins by reversible according to the manufacturer’s instructions. Polyclonal phosphorylation in the flagellum. In another study, flagellum antibodies against the C-terminal part of CK1 (amino acids phosphoproteins were examined during flagella shortening. 131–333 out of 333; ID JGI Vs3: 137286) were also used In this case, postincubation with ATP was undertaken. [23]. For this, the C-terminal part of CK1 was expressed Thereby, half of the identified phosphoproteins were only and purified from E. coli according to the Qiagen protocol. detected in shortening flagella [12]. Antibodies were raised by the “Pineda-Antikor ¨ per-Service,” The axoneme of Chlamydomonas flagella anchors mul- Berlin, Germany. Immunoblots were done as described [11] tiple inner arm dyneins and a network of kinases and using the polyclonal anti-CK1 antibody in a dilution of phosphatases that control motility by reversible protein 1 : 5,000. phosphorylation [13]. One of the involved flagellum kinases is Casein Kinase 1 (CK1) [14–16]. In pharmacological exper- 2.3. Densitometry Analysis. Quantifications were done with iments using a specific CK1 inhibitor (CKI-7), it was shown the Image Master 2D Elite (version 4.01) software from GE that CK1 regulates dynein activity and flagellum motility by Healthcare (formerly Amersham Pharmacia Biotech). phosphorylation of the Inner Dynein Arm I1 Intermediate Chain 138 (IC138) [14, 15]. Moreover, silencing of CK1 2.4. Measurement of Swimming Velocity of C. reinhardtii results in alterations of circadian phototaxis (shortening of Cells. Measurement of swimming velocity was done by using the period), defects in flagella formation, and in hatching a hemocytometer and a differential interference contrast of the daughter cells [17]. Interestingly, alterations in the microscope with a total magnification of 400 including a expression of several other key players of the clock machinery personal computer with a digital video recording system to of C. reinhardtii named Rhythm of Chloroplast (ROC) and measure displacement versus time. The swimming velocity a homologue of Constans (CrCO) have in parallel severe was determined manually by measuring the linear displace- effects on hatching, flagella formation, and/or movement, ment of cells on the scale of the micrometer. 10 samples were underlining that these processes are interconnected in C. measured to obtain the average velocity of a given sample. reinhardtii [17–19]. Regarding the multiple functions of CK1 in flagella 2.5. Cell Growth, CKI-7 Treatment, Isolation of Flagella, formation and motility along with its regulatory role in the Protein Digestion, and Enrichment of Phosphopeptides by circadian system in C. reinhardtii, we were interested in the Immobilized Metal Affinity Chromatography (IMAC). Cells identification of CK1 targets in flagella beside IC138. In a were growninaLD cycleand harvestedatthe endof comparative phosphoproteomic approach using wild-type −1 the night (LD24) at a cell density of 2-3 × 10 cells mL cells with and without CKI-7 treatment, we determined the by centrifugation (700×g, 5 min, 4 C).Cells were resus- targets of CK1 in the flagellum. In the CKI-7-treated cells, pended in one-half volume of minimal medium [21]and several phosphoproteins were missing or were identified with then the culture was kept under dim light conditions for a reduced number of phosphorylation sites, compared to 29 h representing subjective day (LL29), before cells were untreated wild-type cells. Also novel phosphopeptides or harvested (700×g, 15 min, 4 C). In some cases, the CK1 additional phosphorylation sites of known phosphopeptides inhibitor, CKI-7, (N-(2-Aminoethyl)-5-chloroisoquinoline- were identified in the CKI-7-treated cells, suggesting that 8-sulfonamide; Toronto Research Chemicals Inc.) [24], was CK1 is part of a signaling network in the flagellum. added to the culture to a final concentration of 50 μM following the shift to LL conditions. Isolation of the 2. Materials and Methods matrix membrane axoneme fraction (MMA) of flagella, tryptic digestion of MMA proteins, and enrichment of Standard molecular biology methods were done according to flagellum phosphopeptides by IMAC were done as previously [20]. described [11]. 2.1. Cell Culture. C. reinhardtii strain 137c (nit1 nit2)was 2.6. Peptide Identification by Nano-Liquid Chromatography- used with whom the flagellar proteome and phosphopro- Electrospray Ionization-Mass Spectrometry (nLC-ESI-MS) teome were analyzed [4, 11]. Cells were grown in TAP and Data Analysis. nLC-ESI-MS and data analysis were medium [21] under a 12 h light-12 h dark cycle (LD 12 : 12) carried out as described before [11]. Briefly, phosphopep- −2 −1 with a light intensity of 71 μEm sec (1 E = 1mol of tides were subjected to nLC-ESI-MS using an UltiMate photons) at 24 C. The beginning of the light period is 3000 nano-HPLC (Dionex Corporation) with a flow rate of −1 defined as time zero (LD0) and the beginning of the dark 300 nL min coupled online with a linear ion trap ESI-MS period is LD12. In some cases, cells were released after (Finnigian LTQ, Thermo Electron Corp.). The instrument growth in LD into constant conditions (LL) of dim light was run by the data-dependent neutral loss method, cycling −2 −1 (15 μEm sec ). between one full MS and MS/MS scans of the four most International Journal of Plant Genomics 3 abundant ions. After each cycle, these peptide masses were CK1 seems to lead to the activation of other kinases resulting excluded from the analysis for 10 sec. The detection of a in the phosphorylation of other proteins. neutral loss fragment (98, 49, or 32.66 Da) in the MS scans As mentioned before, flagellum kinases affect motility. triggered an MS scan of the neutral loss ion representing the We also studied if the inhibition by CKI-7 results in changes dephosphorylated peptide. in swimming velocity. To analyze the swimming behavior, we compared the swimming velocity of the C. reinhardtii strain Data analysis was done using the Proteome Discoverer 137c with cells that were cultivated with CKI-7 as described software (Version 1.0) from Thermo Electron Corp. includ- (see Section 2). Cells were spotted on a hemocytometer and ing the SEQUEST algorithm [25]. The software parameters the swimming velocity was measured using a differential were set to detect a modification of 79.96 Da in Ser, 2 3 interference contrast microscope including a personal com- Thr, or Tyr in MS and MS spectra. For the database puter with a video recording system (see Section 2). The searches with MS data, modifications of −18.00 Da on assay revealed that the swimming speed of CK1-inhibited Ser and Thr residues representing the neutral loss were cells is significantly lower (75.6 μm/s; ±4,1 SEM) compared additionally used. Further, detection of a modification of to untreated cells (122.2 μm/s; ±2.5 SEM) (Figure 1(d)). 16 Da on Met representing its oxidized form was enabled These data show that CK1-mediated phosphorylation events and carboxyamidomethylation of Cys residues was enabled in flagella influence motility and swimming speed of C. as a static modification. Peptide mass tolerance was set to 2 3 reinhardtii cells. 1.5 Da in MS mode. In MS and MS modes, fragment ion tolerance was set up to 1 Da. The parameters for all 3.2. The Flagellum Phosphoproteome of CKI-7-Treated Cells. database searches were set to achieve a false discovery rate The targets of CK1 in the flagellum are of high interest with (FDR) of not more than 1% for each individual analysis. regard to flagella formation as well as for clock control events. Data were searched against the flagellar proteome database They are largely unknown. An exception is IC138 that is [4](http://labs.umassmed.edu/chlamyfp/index.php). Addi- suggested as a direct target of CK1 based on experimental tionally, NCBI and the Joint Genome Institute C. reinhardtii data (summarized in [25]). databases (Version 2 and Version 3) were used for data In a next step, the direct and indirect targets of CK1 evaluation. were analyzed by a functional proteome approach. For that purpose, we compared the already existing phosphopro- 3. Results teome [11] with one investigated exactly under the same conditions with the single exception that CK1 is inhibited. 3.1. The Effects of the CK1 Inhibitor CKI-7 on the Phos- Since strong silencing of CK1 by RNAi results in defects phorylation Pattern of Flagellum Proteins and the Swimming in flagella formation, flagellum material cannot be obtained Velocity of C. reinhardtii. CK1 was found in the proteome in a significant amount from such strains [17]. Therefore, of the flagellum [4] and was also shown immunologically to inhibition of CK1 with CKI-7 was used. Cells were grown be enriched in flagella in wild-type strain SAG 73.72 [17]. under a light-dark cycle and the inhibitor was added for a For the comparative phosphoproteome analysis, flagella were 29 h period right at the moment when the cells were released isolated from strain 137c along with the dibucaine method to constant dim light. LL29 was also used as harvesting time [11]. We first examined the enrichment of CK1 in flagella point in the previous analysis [11]. of 137c using the applied conditions by immunodetection We avoided to add high amounts of ATP to isolated along with anti-CK1 antibodies (Figures 1(a) and 1(b)). Lev- flagella and to postincubate them at elevated temperatures els of CK1 were significantly enriched in the flagella fraction, to induce kinase activities in vitro, as done in another especially compared to cell bodies lacking flagella. Thus, the study [12]. We found that this treatment leads to severe procedure used for identification of the phosphoproteome phosphorylation events that include most likely phospho- maintains the enrichment of CK1 in flagella and is thus rylation steps that would not take place in vivo under suited to screen for its targets. physiological conditions See Supplemental Figures 1(a), In the next step, we examined to what degree the 1(b) in Supplementary Material available online at doi: CK1-specific inhibitor, CKI-7 [24], which was already used 1155/2012/581460. for studying CK1 in C. reinhardtii [15], influences the The further analysis of the phosphoproteome in CKI-7- phosphorylation pattern of flagellum proteins. Therefore, we treated cells was carried out with the same procedure and grew cells with and without CKI-7 treatment, respectively, criteria as applied before for the flagellum phosphoproteome and compared the flagellum phosphoproteins from both [11]. Three independent isolations of flagella of CKI-7- aliquots by immunodetection with antiphosphoSer anti- inhibited cells were carried out and subjected to phospho- bodies (Figure 1(c)). As expected, several phosphorylated peptide purification along with liquid chromatography mass protein bands were reduced to a significant extent or spectrometry (for details, see [11]). Previously identified absent in the CKI-7-inhibited cells (Figure 1(c), labeled with phosphopeptides or phosphorylation sites within a phospho- “−”). At the same time, some phosphoprotein bands were peptide ( listed in Table S1 in [11]) that had not been detected stronger (Figure 1(c), labeled with “+”). These data show in any of the three analyses were considered to be either direct that inhibition of CK1 has a dual effect. On the one hand, or indirect targets of CK1. The phosphoproteins to which the phosphorylation of CK1 targets drops strongly down or these phosphopeptides belong are listed in Table 1.Novel is fully stopped by its inhibition; on the other hand, inactive phosphopeptides belonging to novel phosphoproteins that 4 International Journal of Plant Genomics CK1 CE CB FL CE CB FL (a) (b) CKI-7 − + − 60 −CKI-7 +CKI-7 (c) (d) Figure 1: Enrichment of CK1 in flagella and the influence of CK1 inhibition on the phosphorylation status of flagellum proteins and swimming velocity of C. reinhardtii cells. (a) Cells were grown in TAP in a 12 h light-12 h dark cycle and then released to dim light (LL) according to Section 2. Cells were harvested at LL29 and flagella were isolated and a whole cell crude extract (CE), a flagellar extract (FL), and an extract from cell bodies lacking flagella (CB) were prepared. 25 μg proteins per fraction were separated by SDS-PAGE and analyzed by immunoblotting with anti-CK1 antibodies according to Section 2. (b) For quantifications, the amount of CK1 detected in the whole cell crude extract was set to 100%. Quantifications were done with three biological replicates using the ImageMaster 2D Elite Vs.4.01 software (GE Healthcare). (c) Changes in the phosphorylation pattern of flagellum proteins in cells treated with and without CKI-7. Cells were grown as described above (a) in the presence or absence of CKI-7 and harvested at LL29 before isolation of flagella. Proteins from the MMA fraction of the flagellum (25 μg each lane) were separated by 9% SDS-PAGE along with a molecular mass standard and immunoblotted with specific antibodies against phosphoSer according to Section 2. Changes in the phosphorylation status of proteins after CKI-7 treatment are indicated by “+” and “−” signs, respectively. (d) Swimming velocity of 137c cells in the absence (−CKI-7) or presence of CK1 inhibitor (+CKI-7). Cells were grown at 23 C in a LD cycle. Measurements of swimming velocity were done with a hemocytometer and a differential interference contrast microscope with a total magnification of 400 including a personal computer with a video recording system to measure displacement versus time (n = 10). Error bars represent the SEM. had not been identified in the former analysis and additional different combinatory phosphorylation patterns (data not phosphopeptides or phosphorylation sites of already identi- shown). fied phosphoproteins are listed in Table 2. Details about all In the CKI-7-treated cells, phosphopeptides from 14 newly identified peptides and phosphorylation sites can be phosphoproteins were missing (Table 1). Four additional found in Supplemental Table S1. In three cases, (TEKTIN, phosphoproteins were identified again but with a reduced FAP18, and FAP262), all previous identified phosphorylation number of phosphorylation sites. These are labeled by sites were detected again, but in some phosphopeptides with indices along with the missing sites in Table 1.Among (kDa) Swimming velocity (µm/s) Relative abundance (%) International Journal of Plant Genomics 5 KLP1 PF6 C1 C2 IFT43 H RSP11 RSP17 IC138 DC2 DC1 (a) RSP11 0 204 RIIa 30 31 32 33 34 35 36 37 38 39 40 41 RQ P Tp DL I A F SA K (b) GSK3 Ser/Thr kin 231 232 233 234 235 236 237 238 239 240 241 242 243 244 I I CS R L KE G P N SYp L KE G P NI Sp Y I CS R (c) CK1 Kinase GSK3 (active) (inactive) (inactive) (d) CKI-7 GSK3 CK1 Kinase (active) (active) (inactive) (e) Figure 2: Analysis of CK1 targets in the flagellum. (a) Diagram of flagellum phosphoproteins in wild-type and CK1-inhibited cells. A cross-section of a flagellum from C. reinhardtii (left panel) and a more detailed view (red rectangle) are shown according to [11]. Structural phosphoproteins in CKI-7-inhibited cells, and such with a reduced number of phosphorylation sites are indicated in yellow color with a red frame. Novel phosphopeptides of structural proteins or additional phosphorylation sites of known phosphopeptides from structural phosphoproteins that were identified in the CKI-7-inhibited proteome are indicated by yellow color with a blue frame. Structural proteins with previously identified phosphopeptides, whose phosphorylation sites were detected again, are indicated in yellow without frame. Abbreviations are: C1 central pair projection (C1P), C2 central pair projection (C2P), PF6 protein (PF6), Hydin (H), Radial Spoke Protein17 (RSP17); Outer Dynein Arm Docking Complex (DC); Inner Dynein Arm Intermedite Chain138 (IC138), Tectin (T) as well as an Intraflagellar Transport Protein43 (IFT43). (b) and (c) Positions of identified phosphopeptides in the predicted domains of RSP11 (b) and GSK3 (c). Identified phosphopeptides are indicted by black boxes. The amino acid positions are mentioned. “p” indicates in vivo phosphorylation sites. RIIa, regulatory subunit of cAMP-Dependent Protein Kinase A; Ser/Thr Kin, Ser/Thr protein kinase catalytic domain. (d) and (e) Hypothetical model of GSK3 de-/activation via reversible phosphorylation triggered by CK1. (d) Regulatory signaling involves an additional kinase. The noninhibited active form of CK1 inactivates another still unknown kinase by phosphorylation. This kinase is needed in its active nonphosphorylated form for activating GSK3. (e) If CK1 is inhibited by CKI-7, the unknown kinase is not phosphorylated and thus active. This active kinase phosphorylates in turn GSK3, which is then activated. 6 International Journal of Plant Genomics Table 1: Phosphoproteins identified in 137c [11] whose phospho- Table 2: Additional phosphopeptides/phosphorylation sites in peptides or phosphorylation sites are missing in CKI-7-treated cells. CKI-7-treated cells of either novel phosphoproteins or phosphopro- teins that were already identified in 137c [11]. Flagellar central pair-associated protein; PF6 Phosphoproteins only present in CKI-7-treated 137c cells Hydin-like protein; HYD3 Glycogen synthase kinase 3; GSK3 Inner dynein arm I1 intermediate chain; IC138 Kinesin-like protein; Kinesin motor domain, KIF9-like subgroup Intraflagellar transport protein IFT43 a,b Phosphoglucomutase Outer dynein arm docking complex subunit 1 ;ODA-DC1, ODA3 Radial spoke protein 11; RSP11; RIIa domain Radial spoke protein 17; RSP17 S-Adenosylmethionine synthetase FAP59 ; RecF/RecN/SMC N-terminal domain FAP139 ; TIGR02680 domain a,d,e FAP116 ; microtubule-binding protein MIP-T3 domain FAP21 a,f FAP190 ; sterile alpha motif FAP56 FAP228; callose synthase-like protein; 1,3-beta-glucan synthase FAP75 component FAP98 FAP230; ankyrin repeats; ion transport protein domain FAP129 FAP254; putative ankyrin-like protein FAP165 FAP288; EF hand FAP236 a,g FAP1 FAP241 FAP93 FAP243 (Vs3 FAP183) FAP147 Phosphoproteins found in CKI-7-treated cells with additional FAP184 phosphopeptide(s) in comparison to 137c FAP263 Outer dynein arm docking complex protein 2; ODA-DC2 The function of depicted proteins is given as determined by NCBI BLASTp, a,b FAP33 ; ankyrin repeats along with their conserved domains. FAP154 Not all previously identified peptides (listed in Table S1 in [11]) are present in the CKI-7-treated cells. FAP217 Variants of peptide TISGADTPEEVLAYWEGLK with the phosphorylation Phosphoproteins found in CKI-7-treated cells with the same sites Thr-345, Ser-347, and Thr-351 as well as variants of peptide ILGYTGS- peptide [11] but with additional phosphorylation site(s) DVEEEEPESEEETEEEANKDDGVVDR with the phosphorylation sites Tyr- 697 and Ser-709 are missing. FAP39 ; plasma membrane calcium transporting ATPase Predicted functional domains are present only in the Vs3 model. d MAK7 ; mitogen activated protein kinase 7 Vs2 model differs significantly from Vs3 model. The phosphorylation site Ser-255 in peptide SASPGGEDPLNKSGSAAPK The function of depicted proteins is given as determined by NCBI BLASTp, is missing. along with their conserved domains. Variants of peptide STSSIGGGYSEPVGSDGEGSDAASAKPR with phos- Vs2 model differs significantly from Vs3 model. phorylation sites on Ser-370, Ser-375 and Ser-379 are missing. Predicted functional domains are present only in the Vs3 model. The phosphorylation site Ser-55 in peptide SRGSFQEGQAMVR is missing. Protein Kinase A (PKA) and bears a phosphorylation site these 18 phosphoproteins, six known structural proteins are (Figure 2(b)). Two other kinases were also found in this present including IC138 that was suggested to be a direct category. One of them is Glycogen Synthase Kinase 3 (GSK3). target of CK1 [26]. All missing structural phosphoproteins as The level of active GSK3 is postulated to be regulated via well as those with a reduced number of phosphorylation sites phosphorylation of a conserved Tyr correlating with flagellar are indicated in yellow color with a red frame in Figure 2(a). length [27]. Exactly this Tyr that is situated in the Ser/Thr Moreover, seven FAPs with conserved domains are affected kinase domain of GSK3 is phosphorylated as well as a in the CKI-7-treated phosphoproteome as well as five FAPs Ser in its surroundings (Supplemental Table 1; Figure 2(c)). without any conserved domains. Notable GSK3 is also clock relevant, for example, in Also novel phosphopeptides or additional phosphory- Drosophila [28]. Moreover, a Mitogen Activated Kinase, lation sites of known phosphopeptides were identified in MAK7, was found with additional phosphorylation sites. the proteome of CKI-7-treated cells, suggesting that CK1 is part of a signaling network in the flagellum. They belong 4. Discussion to either 15 new phosphoproteins or six already known phosphoproteins (Table 2, Supplemental Table 1). Among The identification of targets of CK1 in the flagellum will them, some structural components are present, indicated by help understanding flagella formation as well as clock yellow color with a blue frame in Figure 2(a).Thereby,Radial control events related to flagella [17–19]. The fact that Spoke Protein 11 (RSP11) is of special interest. It has an RIIa several phosphorylated flagellar protein bands disappear in domain, which is a regulatory subunit of cAMP Dependent CKI-7-treated cells suggests that CK1 has multiple targets International Journal of Plant Genomics 7 in the flagellum. Among the 32 phosphoproteins of the was reduced to a similar degree in comparison to the flagellum, 14 were missing in the flagellum phosphopro- mutant strains that are lacking IC138, suggesting that the teome when the CKI-7 inhibitor was used or represented generation of flagellum motility is regulated by a CK1- with a reduced number of phosphorylation sites (four mediated phosphorylation of IC138 as suggested before [14, cases, Table 1). Missing phosphorylation sites cannot be 15]. automatically considered as direct targets of CK1. It could Another structural phosphoprotein previously identified be that the phosphorylation of an amino acid residue by with two phosphopeptides and variable phosphorylation CK1 represents a trigger that then allows a consequent sites is ODA-DC1. The outer dynein arm docking complex phosphorylation of another amino acid residue in the (ODA-DC), which is composed of three proteins, designated surroundings by another kinase. An example for consequent DC1, DC2, and DC3, is associated with microtubules phosphorylation steps of different kinases is mentioned and targets the outer dynein arms to its binding site on below and involves PKA, GSK3, and CK1. Also, CK1 may the flagellum axoneme [33]. In both previously identified activate or deactivate another kinase by reversible phos- phosphopeptides certain phosphorylation sites are missing phorylation. In the current study, the previously identified in CKI-7-inhibited cells (Table 1; indices a, b) pointing out kinases along with their phosphorylation sites were found that they are CK1 targets. ODA-DC2 had been also identified again [11]. Only in case of FAP262 that bears a Ser/Thr kinase in the previous study [11] with one phosphopeptide and domain, a different combinatory phosphorylation pattern variable phosphorylation sites, which were all found again was observed, which might be relevant. But it could also in the current study. But now a novel phosphopeptide with be that some of the missing phosphoproteins in the FAP phosphorylation on Ser-278 was present in CKI-7 cells, category whose functions are not known may have kinase underlining that CK1 seems to be indirectly involved in activity. Networks that consist of interconnected kinases regulating further kinases. along with protein phosphatases are not unusual in signaling. Radial spokes represent a major structural feature of 9+2 In line with this, we found also 21 new phosphoproteins axonemes and they are essential for flagellum beating. Each along with novel phosphopeptides or phosphorylation sites, radial spoke consists of a thin stalk, which is attached to including three kinase-related proteins. The presence of new the A-tubule of the axonemal doublet microtubules and a phosphorylation sites in flagella of CKI-7-inhibited cells head projecting toward the central apparatus [34]. The radial was already predictable from the appearance of novel flag- spoke of C. reinhardtii is composed of at least 23 proteins, ellar phosphoprotein bands detected with anti-phosphoSer and not all of them have been characterized at the molecular antibodies (Figure 1(c)). In this category, we identified level [35]. RSP17, which is located in the spoke stalk, two phosphoproteins involved in carbohydrate and amino was identified in the flagellum phosphoproteome analysis acid metabolism, respectively (Table 2). One of them, with two different phosphopeptides [11]. The absence of phosphoglucomutase, catalyzes the bidirectional conversion both phosphopeptides in CKI-7-treated cells suggests that of glucose-1-phosphate to glucose-6-phosphate. Glucose-1- RSP17 is at the same time a direct and/or indirect target phosphate can be transferred into glycolysis by this way. The of CK1. Functional domains in radial spoke proteins reveal flagellum contains all enzymes of the late glycolytic pathway; their role in mediating signaling pathways. For instance, they are able to generate ATP for direct use in the flagellum RSP11 consists of a regulatory subunit (RIIa) of PKA [35]. [4]. In mammals, the activity of phosphoglucomutase is However, RSP11 lacks the cAMP-binding domains of the RII regulated by phosphorylation [29]. The other metabolically regulatory subunit. We could identify RSP11 in the CKI- relevant enzyme in this category is S-adenosylmethionine 7-treated cells as a new phosphoprotein with one in vivo synthetase, a key enzyme of methionine metabolism. In rat phosphorylation site at Thr-35, which is located directly liver, the activity of the S-adenosylmethionine synthetase is in the RIIa domain (Figure 2(b)). The interaction between regulated by Protein Kinase C [30]. RII and A-kinase anchoring protein motifs (AKAP) can be One of the direct targets of CK1 was suggested to be regulated by phosphorylation of RII [36, 37]. A pharma- IC138, the Inner Dynein Arm I1 Intermediate Chain 138. cological analysis using an inhibitor and the RII regulatory It was shown that phosphorylation of IC138 correlates with subunits had detected an axonemal PKA activity [38]. But the inhibition of dynein activity and that PKA beside CK1 as PKA could not be found in the flagellar proteome in contrast well as the Protein Phosphatases PP2A and PP1 are involved to CK1, PP1, and PP2A [4]. Thus, it was hypothesized that there (summarized in [26]). IC138 was identified in CK1 C. reinhardtii could express a PKA with an unconventional active cells with one phosphopeptide that is situated at its structure [39]. The identified phosphorylation site within the N-terminus including variable phosphorylation sites [11]. RII subunit of RSP11 may be relevant in this context. None of these phosphorylation sites were detected after An additional flagellum kinase is GSK3 whose enzymatic CKI-7 treatment, underlining that IC138 is a direct and/or activity is inhibited by lithium causing flagellar elongation indirect target of CK1. A pharmacological analysis using [27]. It is known that GSK3 has a Tyr-phosphorylated, active CKI-7 revealed the impact of CK1 on IC138 phosphorylation form and is enriched in flagella. GSK3 is associated with [14]. This mechanism authorizes CK1 to regulate dynein the axoneme in a phosphorylation-dependent manner. The activity and control flagellum motility. Also an analysis of levelofactiveGSK3correlateswithflagellarlength[27]. We mutants lacking the IC138 subcomplex revealed strains that could identify the Tyr-240-phosphorylated GSK3 as well as swim forward with reduced swimming velocities [31, 32]. a Ser-239-phosphorylated alternative in the CKI-7-treated Interestingly, the swimming speed of the CKI-7-treated cells cells (Figure 2(c)), suggesting that inhibition of CK1 causes 8 International Journal of Plant Genomics activation of GSK3. Both in vivo phosphorylation sites are [3] S. S. Merchant, S. E. Prochnik, O. Vallon et al., “The Chlamydomonas genome reveals the evolution of key animal located in the catalytic kinase domain, which could play and plant functions,” Science, vol. 318, no. 5848, pp. 245–251, important roles in the regulation of the activity of GSK3 within signaling pathways. Notably, interplay between CK1 [4] G. J. Pazour, N. Agrin, J. Leszyk, and G. B. Witman, “Pro- and GSK3 is known for Hedgehog signaling pathways [40]. teomic analysis of a eukaryotic cilium,” The Journal of Cell Thereby, the Cubitus Interruptus (Ci-155) transcriptional Biology, vol. 170, no. 1, pp. 103–113, 2005. activator is involved. Ci-155 proteolysis depends on phos- [5] W. F. Marshall, “The cell biological basis of ciliary disease,” The phorylation at three sites of PKA. Then, these phosphoSer Journal of Cell Biology, vol. 180, no. 1, pp. 17–21, 2008. prime further phosphorylation at GSK3 and CK1 sites. This [6] J. Reinders and A. Sickmann, “Modificomics: posttranslational principle is a good example for consecutive phosphorylation modifications beyond protein phosphorylation and glycosyla- steps of different kinases as mentioned before. tion,” Biomolecular Engineering, vol. 24, no. 2, pp. 169–177, Several studies have shown that reversible phosphoryla- tion of Tyr causes increases and decreases in GSK3 kinase [7] N. Rolland, A. Atteia, P. Decottignies et al., “Chlamydomonas activity, respectively [41, 42]. For the interplay of CK1 proteomics,” Current Opinion in Microbiology, vol. 12, no. 3, and GSK3 in the C. reinhardtii flagella, one can imagine pp. 285–291, 2009. a regulatory mechanism, involving, for example, an addi- [8] V. Wagner, J. Boesger, and M. Mittag, “Sub-proteome analysis tional kinase. In a hypothetical model (Figure 2(d)), the in the green flagellate alga Chlamydomonas reinhardtii,” Jour- noninhibited, active CK1 inactivates another kinase by nal of Basic Microbiology, vol. 49, no. 1, pp. 32–41, 2009. phosphorylation, which is responsible for the activation [9] A. V. Vener, “Environmentally modulated phosphorylation of GSK3 by Tyr-phosphorylation. If CKI-7 inhibits CK1 and dynamics of proteins in photosynthetic membranes,” Bio- (Figure 2(e)), the additional kinase can stay active, because chimica et Biophysica Acta, vol. 1767, no. 6, pp. 449–457, 2007. it is not phosphorylated by CK1 and consequently GSK3 gets [10] V. Wagner, K. Ullmann, A. Mollwo, M. Kaminski, M. Mittag, and G. Kreimer, “The phosphoproteome of a Chlamydomonas converted to the phosphorylated active form. reinhardtii eyespot fraction includes key proteins of the light GSK3 plays also an important role in the regulation signaling pathway,” Plant Physiology, vol. 146, no. 2, pp. 772– of circadian systems. Shaggy (SGG), for example, the 788, 2008. Drosophila homologue of GSK3, is a central player in deter- [11] J. Boesger, V. Wagner, W. Weisheit, and M. Mittag, “Analysis of mining period length in flies by phosphorylation of clock flagellar phosphoproteins from Chlamydomonas reinhardtii,” components [43]. In mammals, GSK3 is proposed to phos- Eukaryotic Cell, vol. 8, no. 7, pp. 922–932, 2009. phorylate Clock (CLK), which is a core transcription factor [12] J. Pan, B. Naumann-Busch, L. Wang et al., “Protein phospho- that is essential for circadian behavior. Phosphorylation of rylation is a key event of flagellar disassembly revealed by anal- CLK controls its activity and degradation [44]. Especially ysis of flagellar phosphoproteins during flagellar shortening in kinases and phosphatases, which are relevant in regulating Chlamydomonas,” Journal of Proteome Research, vol. 10, no. 8, circadian clocks in other organisms, are well conserved in pp. 3830–3839, 2011. Chlamydomonas [45]. Interestingly, many output rhythms [13] M. E. Porter and W. S. Sale, “The 9 + 2 axoneme anchors that can be measured like phototaxis, chemotaxis, and multiple inner arm dyneins and a network of kinases and stickiness to glass and mating during the cell cycle involve phosphatases that control motility,” The Journal of Cell Biology, flagella. It is remarkable that kinases like CK1 or GSK3 as vol. 151, no. 5, pp. F37–F42, 2000. well as phosphatases like PP1 and PP2A are physically located [14] P. Yang and W. S. Sale, “Casein kinase I is anchored on in the axoneme [4, 26]. This underlines the important axonemal doublet microtubules and regulates flagellar dynein regulatory function of these components in the flagellum phosphorylation and activity,” The Journal of Biological Chem- istry, vol. 275, no. 25, pp. 18905–18912, 2000. regarding circadian rhythms. [15] A. Gokhale, M. Wirschell, and W. S. Sale, “Regulation of dynein-driven microtubule sliding by the axonemal protein Acknowledgments kinase CK1 in Chlamydomonas flagella,” The Journal of Cell Biology, vol. 186, no. 6, pp. 817–824, 2009. The authors thank the Joint Genome Institute (JGI) in the [16] M. Wirschell, R. Yamamoto, L. Alford, A. Gokhale, A. Gaillard, USA and the Kazusa Institute in Japan for the free delivery of and W. S. Sale, “Regulation of ciliary motility: conserved EST and genome sequences. This study was supported by the protein kinases and phosphatases are targeted and anchored in Deutsche Forschungsgemeinschaft (Grants Mi 373 to MM) the ciliary axoneme,” Archives of Biochemistry and Biophysics, and the BMBF (Project GoFORSYS, Grant no. 0315260A, vol. 510, no. 2, pp. 93–100, 2011. work package to MM). [17] M. Schmidt, G. Geßner, M. Luff et al., “Proteomic analysis of the eyespot of Chlamydomonas reinhardtii provides novel insights into its components and tactic movements,” The Plant References Cell, vol. 18, no. 8, pp. 1908–1930, 2006. [18] T. Matsuo, K. Okamoto, K. Onai, Y. Niwa, K. Shimogawara, [1] G. J. Pazour and G. B. Witman, “The vertebrate primary and M. Ishiura, “A systematic forward genetic analysis iden- cilium is a sensory organelle,” Current Opinion in Cell Biology, tified components of the Chlamydomonas circadian system,” vol. 15, no. 1, pp. 105–110, 2003. Genes and Development, vol. 22, no. 7, pp. 918–930, 2008. [2] J. L. Rosenbaum and G. B. Witman, “Intraflagellar transport,” [19] G. Serrano, R. Herrera-Palau, J. M. Romero, A. Serrano, G. Nature Reviews Molecular Cell Biology, vol. 3, no. 11, pp. 813– Coupland, and F. Valverde, “Chlamydomonas CONSTANS 825, 2002. International Journal of Plant Genomics 9 and the evolution of plant photoperiodic signaling,” Current [36] G. Keryer,Z.Luo,J.C.Cavadore, J. Erlichman, andM. Biology, vol. 19, no. 5, pp. 359–368, 2009. Bornens, “Phosphorylation of the regulatory subunit of type [20] J. Sambrook andD.W.Russel, Molecular Cloning: A Laboratory IIβ cAMP-dependent protein kinase by cyclin B/p34(cdc2) Manual, Cold Spring Harbor Laboratory Press, Cold Spring kinase impairs its binding to microtubule-associated protein Harbor, NY, USA, 2001. 2,” Proceedings of the National Academy of Sciences of the United [21] E. H. Harris, The Chlamydomonas Sourcebook,Academic States of America, vol. 90, no. 12, pp. 5418–5422, 1993. Press, San Diego, Calif, USA, 1989. [37] S. Manni, J. H. Mauban, C. W. Ward, and M. Bond, “Phospho- [22] V. Neuhoff, K. Philipp, H. G. Zimmer, and S. Mesecke, “A rylation of the cAMP-dependent protein kinase (PKA) reg- simple, versatile, sensitive and volume-independent method ulatory subunit modulates PKA-AKAP interaction, substrate for quantitative protein determination which is independent phosphorylation, and calcium signaling in cardiac cells,” The of other external influences,” Hoppe-Seyler’s Zeitschrift fur ¨ Journal of Biological Chemistry, vol. 283, no. 35, pp. 24145– Physiologische Chemie, vol. 360, no. 11, pp. 1657–1670, 1979. 24154, 2008. [23] T. Schulze, S. Schreiber, D. Iliev et al., “The heme-binding [38] A. R. Gaillard, L. A. Fox, J. M. Rhea, B. Craige, and W. S. Sale, protein SOUL3 of Chlamydomonas reinhardtii influences size “Disruption of the A-kinase anchoring domain in flagellar and position of the eyespot,” Molecular Plant. In press. radial spoke protein 3 results in unregulated axonemal cAMP- [24] F. Preuss, J. Y. Fan, M. Kalive et al., “Drosophila doubletime dependent protein kinase activity and abnormal flagellar mutations which either shorten or lengthen the period of motility,” Molecular Biology of the Cell, vol. 17, no. 6, pp. 2626– circadian rhythms decrease the protein kinase activity of 2635, 2006. casein kinase I,” Molecular and Cellular Biology, vol. 24, no. [39] E. H. Harris, The Chlamydomonas Sourcebook, vol. 3, Aca- 2, pp. 886–898, 2004. demic Press, San Diego, Calif, USA, 2009. [25] A. J. Link, J. Eng, D. M. Schieltz et al., “Direct analysis of [40] M. A. Price and D. Kalderon, “Proteolysis of the Hedgehog sig- protein complexes using mass spectrometry,” Nature Biotech- naling effector Cubitus interruptus requires phosphorylation nology, vol. 17, no. 7, pp. 676–682, 1999. by Glycogen Synthase Kinase 3 and Casein Kinase 1,” Cell, vol. [26] M. Wirschell, T. Hendrickson, and W. S. Sale, “Keeping an eye 108, no. 6, pp. 823–835, 2002. on I1:I1 dynein as a model for flagellar dynein assembly and regulation,” Cell Motility and the Cytoskeleton,vol. 64, no.8, [41] L. Kim, J. Liu, and A. R. Kimmel, “The novel tyrosine kinase pp. 569–579, 2007. ZAK1 activates GSK3 to direct cell fate specification,” Cell, vol. 99, no. 4, pp. 399–408, 1999. [27] N. F. Wilson and P. A. Lefebvre, “Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas [42] H. Murai, M. Okazaki, and A. Kikuchi, “Tyrosine dephos- reinhardtii,” Eukaryotic Cell, vol. 3, no. 5, pp. 1307–1319, 2004. phorylation of glycogen synthase kinase-3 is involved in its [28] E. Harms, M. W. Young, and L. Saez, “CK1 and GSK3 in extracellular signal-dependent inactivation,” FEBS Letters, vol. the Drosophila and mammalian circadian clock,” Novartis 392, no. 2, pp. 153–160, 1996. Foundation Symposium, vol. 253, pp. 267–277, 2003. [43] S. Panda, J. B. Hogenesch, and S. A. Kay, “Circadian rhythms [29] A. Gururaj, C. J. Barnes, R. K. Vadlamudi, and R. Kumar, from flies to human,” Nature, vol. 417, no. 6886, pp. 329–335, “Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase,” Oncogene, vol. 23, no. 49, pp. [44] M. L. Spengler, K. K. Kuropatwinski, M. Schumer, and M. 8118–8127, 2004. P. Antoch, “A serine cluster mediates BMAL1-dependent [30] M. A. Pajares, C. Duran, F. Corrales, and J. M. Mato, “Protein CLOCK phosphorylation and degradation,” Cell Cycle, vol. 8, kinase C phosphorylation of rat liver S-adenosylmethionine no. 24, pp. 4138–4146, 2009. synthetase: dissociation and production of an active mono- [45] M. Mittag, S. Kiaulehn, and C. H. Johnson, “The circadian mer,” Biochemical Journal, vol. 303, no. 3, pp. 949–955, 1994. clock in Chlamydomonas reinhardtii. What is it for? What is [31] T. W. Hendrickson, C. A. Perrone, P. Griffin et al., “IC138 it similar to?” Plant Physiology, vol. 137, no. 2, pp. 399–409, is a WD-repeat dynein intermediate chain required for light chain assembly and regulation of flagellar bending,” Molecular Biology of the Cell, vol. 15, no. 12, pp. 5431–5442, 2004. [32] K. E. VanderWaal, R. Yamamoto, K. Wakabayashi et al., “bop5 mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric wave- forms,” Molecular Biology of the Cell, vol. 22, no. 16, pp. 2862– 2874, 2011. [33] S. Takada, C. G. Wilkerson, K. I. Wakabayashi, R. Kamiya, and G. B. Witman, “The outer dynein arm-docking complex: com- position and characterization of a subunit (Oda1) necessary for outer arm assembly,” Molecular Biology of the Cell, vol. 13, no. 3, pp. 1015–1029, 2002. [34] A. M. Curry and J. L. Rosenbaum, “Flagellar radial spoke: a model molecular genetic system for studying organelle assembly,” Cell Motility and the Cytoskeleton,vol. 24, no.4,pp. 224–232, 1993. [35] P. Yang, D. R. Diener, C. Yang et al., “Radial spoke proteins of Chlamydomonas flagella,” JournalofCellScience, vol. 119, part 6, pp. 1165–1174, 2006. International Journal of Peptides Advances in International Journal of BioMed Stem Cells Virolog y Research International International Genomics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Nucleic Acids International Journal of Zoology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific Journal of Signal Transduction World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Advances in Genetics Anatomy Biochemistry Research International Research International Microbiology Research International Bioinformatics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Enzyme Journal of International Journal of Molecular Biology Archaea Research Evolutionary Biology International Marine Biology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Journal

International Journal of Plant GenomicsHindawi Publishing Corporation

Published: Dec 18, 2012

References