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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, email@example.com 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 . 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 . 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 . see ). 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 . C. reinhardtii uses ﬂagella for motility lyzed including environmentally modulated photosynthetic in aqueous environments, for attaching to surfaces and for membranes , the eyespot , and the ﬂagellum . The ﬂagellum phosphoproteome was ﬁrst studied under cell-cell recognition during mating. A proteomic analysis of Chlamydomonas ﬂagella revealed more than 600 proteins  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 . The kinases, and proteins with protein interaction domains concentration of proteins was measured according to . . 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. . 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 . Antibodies were raised by the “Pineda-Antikor ¨ per-Service,” The axoneme of Chlamydomonas ﬂagella anchors mul- Berlin, Germany. Immunoblots were done as described  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 . 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 . 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 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.) , 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 . described . 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  under a 12 h light-12 h dark cycle (LD 12 : 12) carried out as described before . 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 . 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 (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 ). 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  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 . Therefore, of the ﬂagellum  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 . 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 . We ﬁrst examined the enrichment of CK1 in ﬂagella point in the previous analysis . 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 . 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 , which was already used 1155/2012/581460. for studying CK1 in C. reinhardtii , 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 . 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 ). 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 ) 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 . 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  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 . 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 ) 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  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 . 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 . 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 . 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 . 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 . Only in case of FAP262 that bears a Ser/Thr kinase in the previous study  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 . 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 . 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 . 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 . . In mammals, the activity of phosphoglucomutase is However, RSP11 lacks the cAMP-binding domains of the RII regulated by phosphorylation . 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 . 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 . 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 . Thus, it was hypothesized that there (summarized in ). 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 . The identiﬁed phosphorylation site within the N-terminus including variable phosphorylation sites . 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 . 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 . 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. 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  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  G. J. Pazour, N. Agrin, J. Leszyk, and G. B. 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