Protein Phosphorylation Is a Key Event of Flagellar Disassembly

TECHNICAL NOTE pubs.acs.org/jpr

Protein Phosphorylation Is a Key Event of Flagellar Disassembly Revealed by Analysis of Flagellar Phosphoproteins during Flagellar Shortening in Chlamydomonas Junmin Pan,*,†,# Bianca Naumann-Busch,‡,# Liang Wang,†,# Michael Specht,‡,# Martin Scholz,‡ Kerstin Trompelt,‡ and Michael Hippler*,‡ † ‡

Protein Science Laboratory of the Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China Institute of Plant Biochemistry and Biotechnology, University of M€unster, Hindenburgplatz 55, 48143 M€unster, Germany

bS Supporting Information ABSTRACT: Cilia are disassembled prior to cell division, which is proposed to regulate proper cell cycle progression. The signaling pathways that regulate cilia disassembly are not well-understood. Recent biochemical and genetic data demonstrate that protein phosphorylation plays important roles in cilia disassembly. Here, we analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing shortening as well as flagella from steady state cells of Chlamydomonas. The phosphopeptides were enriched by a combination of IMAC and titanium dioxide chromatography with a strategy of sequential elution from IMAC (SIMAC) and analyzed by tandem mass spectrometry. A total of 224 phosphoproteins derived from 1296 spectral counts of phosphopeptides were identified. Among the identified phosphoproteins are flagellar motility proteins such as outer dynein arm, intraflagellar transport proteins as well as signaling molecules including protein kinases, phosphatases, G proteins, and ion channels. Eighty-nine of these phosphoproteins were only detected in shortening flagella, whereas 29 were solely in flagella of steady growing cells, indicating dramatic changes of protein phosphorylation during flagellar shortening. Our data indicates that protein phosphorylation is a key event in flagellar disassembly, and paves the way for further study of flagellar assembly and disassembly controlled by protein phosphorylation. KEYWORDS: phosphoproteomics, cilia, flagella, Chlamydomonas, protein phosphorylation

’ INTRODUCTION Cilia and flagella are ancient cellular structures and conserved through evolution. The first eukaryote is proposed to possess a single cilium or flagellum.1,2 Though this structure is lost in higher plant lineage during evolution, it is well conserved in vertebrate.3 Cilia are present in almost every mammalian cell. They play pivotal roles in regulating cell motility, signal perception, and transduction, and thus control development, physiology, and cell cycle progression. Defects in cilia structure and function have been linked to various human diseases including polycystic kidney disease, male infertility, abnormal animal development, and even cancer.4 6 In the core of a cilium is a microtubule-based axoneme, which is assembled on modified centriole, called basal body. The axoneme is enclosed by ciliary membrane that is continuous with cytoplasmic membrane. In motile cilia, the axoneme includes substructures such as dynein arms and radial spokes, and one pair of central microtubule in addition to the 9 peripheral doublet microtubules, which are essential for ciliary waveform formation and motility.7 The narrow compartment between the r 2011 American Chemical Society

axoneme and the ciliary membrane is called matrix and contains ciliary precursors, turnover products, and protein machinery for intraflagellar transport (IFT), a bidirectional movement of protein complex that is required for cilia assembly, maintenance, and disassembly.8 10 Cilia are dynamic cellular organelles. Though the presence of cilia is necessary for various cellular function, cilia may be resorbed by gradual disassembly starting at the ciliary tip during cell differentiation, in response to cellular stress or prior to cell division.11 14 The ciliary disassembly process involves at least three functional domains: signaling proteins that initiate and regulate the disassembly, effectors that act on the disassembly per se, and transport systems that ferry disassembled components such as tubulin back to the cell body. Our current understanding of ciliary disassembly has been largely attributed to the research in Chlamydomonas, a widely used model organism for cilia study. During flagellar disassembly in Chlamydomonas, Received: May 10, 2011 Published: June 13, 2011 3830

dx.doi.org/10.1021/pr200428n | J. Proteome Res. 2011, 10, 3830–3839

Journal of Proteome Research methylated as well as ubiquitinated flagellar proteins are increased, indicating active control of protein methylation and ubiquitin conjugation system in facilitating flagellar disassembly.15,16 CrKinesin13, a microtubule depolymerizer, is transported to the flagella at the time when flagella are induced to disassemble. Defect in transport of CrKinesin13 impairs flagellar disassembly.17 The disassembly of flagellar components is accompanied by the regulation of the IFT machinery. The trafficking of IFT proteins is increased and cargo-loading at flagellar entrance is suppressed, which is proposed to remove disassembled flagellar components rapidly for recycling while avoiding compromise of the disassembly process.18 Recent biochemical and genetic data demonstrate that protein phosphorylation is a key regulatory event of flagellar disassembly (see review 19). An aurora-like protein kinase, CALK, in Chlamydomonas mediates flagellar disassembly. CALK is rapidly phosphorylated upon induction of flagellar shortening. Protein kinase inhibitor, as well as knockdown of CALK expression by RNAi, also inhibits flagellar shortening.20 It is predicted that CALK phosphorylation is involved in flagellar disassembly. Consistent with this study, aurora A (a homologue of CALK) has been shown to be required for primary cilia disassembly.21 Flagellar length is maintained by a balance between the assembly and disassembly activities. Disruption of either process results in abnormal flagellar length. Two flagellar length mutants lf2 and lf4 are defective in genes encoding protein kinases, a nonconventional CDK and a MAP kinase, respectively.22,23 It is proposed that LF4 functions by inhibiting flagellar disassembly. Genetic manipulation of NIMA and MAP kinases in Tetrahymena, Chlamydomonas, and Leishmania also impairs flagellar disassembly or length.24 26 Identification of the protein substrates of these kinases and their cellular localization would reveal how protein phosphorylation regulates flagellar assembly and disassembly. In vivo and in vitro labeling of flagellar proteins with 32P demonstrates that Chlamydomonas flagella are enriched in phosphoproteins.27,28 An understanding of cilia assembly and disassembly controlled by protein phosphorylation requires identification of phosphoproteins present in the cilia. Phosphoproteomic analysis provides information about the identity of the phosphoprotein as well as the phosphorylated amino acids residue(s). Recently, the flagellar phosphoproteome of Chlamydomonas has been analyzed and a total of 32 flagellar phosphoproteins have been identified,29 where traditional ion metal-chelate affinity chromatography (IMAC) technique was used to enrich phosphopeptides. To determine flagellar disassembly controlled by protein phosphorylation, we have analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing flagellar shortening. The phosphopeptides were enriched by using a strategy of sequential elution from IMAC (SIMAC), which allows detection of more phosphopeptides than the traditional IMAC technique.30 To reveal differential phosphorylation of flagellar proteins, we have also analyzed the counterpart of flagella of steady state cells. A total of 224 proteins derived out of 1296 spectral counts of phosphopeptides have been identified in shortening flagella and steady state flagella. The identified phosphoproteins of disassembling flagella included IFT proteins, protein kinases, and enzymes in protein methylation and ubiquitination pathway.

’ MATERIALS AND METHODS Strains, Cell Culture, and Induction of Flagellar Disassembly

Chlamydomonas reinhardtii strain 21gr (CC-1690) was obtained from Chlamydomonas Genetic Center. Cells were grown

TECHNICAL NOTE

with shaking in Tris-acetate-phosphate medium with a 12/12 h light dark cycle at 22 °C.31 To induce flagellar shortening, cells were treated for 5 min with sodium pyrophosphate (pH 7.2) at a final concentration of 20 mM followed by flagellar isolation.11 Flagella Isolation and Flagellar Images

Flagella were isolated essentially as described.18 Flagella pellets were resuspended in HMDEK buffer (20 mM HEPES [pH 7.2], 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 25 mM KCl, 0.1% Triton X-100 containing Halt protease and phosphatase inhibitor cocktail (cat. no. 78443, Thermo)). Flagella were then incubated at 25 °C for 10 min in the presence of 1 mM ATP to recover any lost phosphate group during flagellar isolation. The membrane/matrix fraction was collected by centrifugation for 10 min at 14 000 rpm and 4 °C in a table-top centrifuge (Beckman), followed by flash-freezing in liquid nitrogen, and storage at 80 °C. Flagella isolated before solubilization in buffer were fixed in 1% glutaraldehyde and phase contrast images were captured with Zeiss microscope with a 40 tmelens (Zeiss, Germany) Western Blot Analysis

Treatment of cell samples and Western blot analysis was essentially as described.17 Rabbit anti-CALK serum was used at 1:2500. Protein Measurement and Trypsin Digestion

The amount of flagellar proteins was determined with a BCA Protein Assay Kit (cat. 23225, Pierce). Absorbance at 562 nm was measured in a UV/visible spectrometer (Ultrospec 3100 pro, Pharmacia) following the manufacturer’s instructions. BSA was used as protein standard. Trypsin digestion of proteins was carried out using sequencing grade modified trypsin (cat. VA11A, Promega) and the protocols provided thereon. Briefly, 1.2 mg of flagellar proteins dried in a vacuum concentrator (Concentrator 5301, Eppendorf) was digested with 20 μg of trypsin at 37 °C for 12 h. Purification and Enrichment of Phosphopeptides by SIMAC and Identification by Tandem Mass Spectrometry

The purification and enrichment of phosphopeptides was carried out according to the SIMAC protocol published recently.30 In short, the tryptic peptides were first incubated with IMAC affinity matrix (Phos-Select Iron Affinity Gel, Cat. no. P9740, Sigma). Afterward, the gel was applied to empty TopTips (Glygen Corp.) and washed with loading buffer. The flow-though and wash fractions were pooled for further processing. Then, mono and multiply phosphorylated peptides were sequentially eluted using acidic and basic eluents, respectively. The phosphopeptides in the flow-through/wash fraction and the monophosphorylated peptide elution were further enriched by TiO2 chromatography (TopTip Titanium dioxide, cat. no. TT3TIO.20, Glygen Corp). Peptides were separated on an Ultimate 3000 nanoflow HPLC system (Dionex) coupled with an LTQ Orbitrap XL mass spectrometer (Thermo Finnigan). The mobile phases used for peptide separation were 5% (v/v; A) and 80% (v/v; B) acetonitrile in ultrapure water/0.1% (v/v) formic acid. Samples were loaded at 25 μL/min onto a trap column (C18 PepMap100, 300 μM 5 mm, 5 μm particle size, 100 Å pore size, LC Packings) and desalted for 30 min using 5% B. Afterward, the trap column was switched into backflush position in series with a separation column (Atlantis dC18, 75 μm 15 mm, 3 μM particle size, 100 Å pore size, Waters) and the following gradient was applied: 5 60% B over 240 min, 60 95% B over 10 min, 3831

dx.doi.org/10.1021/pr200428n |J. Proteome Res. 2011, 10, 3830–3839

Journal of Proteome Research

TECHNICAL NOTE

95% B for 9 min. The column was re-equilibrated with 95% A for 20 min. Flow rate during separation was 300 nL/min. Peptides eluted directly into the nanospray source of the mass spectrometer and were analyzed in positive ion mode. Survey scans were obtained by FT-MS (m/z 400 2000) at a resolution of 60 000 fwhm using internal lock mass calibration on m/z 445.12003. The five most intense ions of each survey scan were sequentially subjected to CID-fragmentation (35% normalized collision energy, 2 Da isolation width) in the linear ion trap (MS2). Fragment ions exhibiting a neutral loss of 98, 49, or 33 Da were activated further, while the remaining MS2 ions were stored (multistage activation, MSA). After the second step of fragmentation, MS2/MS3 ions were simultaneously analyzed in the ion trap. Dynamic exclusion was enabled with a repeat duration of 90 s, repeat count of 1, list size of 500, and exclusion mass width of (5 ppm. Data Analysis

For the identification of peptides, OMSSA (version 2.1.4);32 and SEQUEST (Bioworks, version 3.3.1 SP1, Thermo) were used in parallel. The following search parameters were identical for both algorithms: the maximum number of missed cleavages allowed was 2. Mass accuracy was set to 5 ppm for MS1 precursor ions and 0.8 Da for product ions. Serine, threonine, and tyrosine phosphorylation was used as variable modifications. The database required for peptide identification was created by merging JGI 3.1 and JGI 4 gene models and adding mitochondrial (NC_001638.1) and plastid (BK000554) sequence data obtained from GenBank. The resulting database was further supplemented with flagella proteome sequence data published by Pazour et al.33 After redundancies had been removed from this composite database, decoy proteins were generated by reversal of each protein sequence. OMSSA was integrated into the MS/MS data processing pipeline Proteomatic34 allowing results to be filtered via a target/decoy approach (FDR = 2ndecoys/(ntargets + ndecoys). In addition, the peptide/spectral matches resulting from the OMSSA search were filtered in such a way that if multiple nonphosphorylated peptides were identified for a single scan; the corresponding E-values were required to be at least 2 orders of magnitude worse compared to the best scoring peptide, as described in Specht et al.35 In those cases where multiple peptides were assigned to the same MS/MS scan with similar E-values, all identifications for that scan were discarded. However, if similar scores were caused solely by ambiguous phosphorylation site assignments, corresponding results were considered equivalent and listed accordingly. For each data set consisting of identifications from all SIMAC fractions of an individual protein sample, an adaptive E-value threshold was determined limiting the estimated false discovery rate (FDR) to 1% or lower. The software package Trans-Proteomic Pipeline (version 4.4) served as platform for SEQUEST searches36 and provided PeptideProphet37 for peptide probability scoring. Search results of each SIMAC fraction were validated individually. Minimum peptide probability allowed was 0.97 to ensure that the FDR was 1% or lower.

’ RESULTS AND DISCUSSION Flagellar Isolation, Sample Processing, and Data Analysis

To investigate flagellar disassembly controlled by protein phosphorylation, we started to analyze the phosphoproteins of flagella undergoing flagellar shortening. Chlamydomonas vegetative

Figure 1. Isolation of disassembling flagella and flowchart of phosphoproteomic analysis. (A) Western blot analysis of CALK after treatment with sodium pyrophosphate (NaPPi) to induce flagellar disassembly. Upper form CALK was phosphorylated. (B) Phase contrast images of isolated flagella. Bar, 5 μm. (C) Flowchart of phosphopeptide enrichment and analysis.

cells were induced to shorten their flagella by treatment with sodium pyrophosphate, an established protocol for inducing flagellar disassembly.11 Phosphorylation of protein kinase CALK is an early event of flagellar disassembly. The phosphorylated CALK exhibited molecular weight shift in Western blot analysis.20 Phosphorylation of CALK was detected in whole cells by Western blot analysis after 5 min treatment, indicating that flagella disassembly was induced (Figure 1A). Isolated flagella were examined microscopically and found to contain pure flagella (Figure 1B). The membrane and matrix fractions of the flagella were collected and digested with trypsin. Phosphopeptides are enriched and collected by sequential elution from IMAC (SIMAC), which is advantageous over traditional IMAC.30 A flowchart of 3832



TECHNICAL NOTE

Table 1. Phosphopeptides and Phosphoproteins Identified from Control Flagella and Disassembling Flagellaa control flagella OMSSA SEQUEST All peptides

Modified peptides

OMSSA

211 (961) 253 (1417) 402 (2275) 525 (3865) 316 (1925) 578 (4793) 193 (358)

70 (191)

289 (555)

108 (358)

149 (555)

135 (497) All phosphoproteins Specific phosphoproteins a

133 (326)

4

173001

2

4

182699

3

6 2

150075

2 2

188947

7 2

195 (799)

163048 (Chlre2) 332365 (Chlre4)

1

4 3

89 (799)

142514

2

4

144131

3

189281

1

224 (1296) 29 (497)

8 2 2

144026

108 (326)

D

410161 (Chlre4) 142470

191579

327 (799)

55 (191)

C

187000

SEQUEST

1877 (4793)

232 (497) Modified proteins

ID

disassembling flagella

465 (961) 530 (1417) 1156 (2275) 1512 (3865) 752 (1925)

All proteins

Table 2. “Unkown” or “Hypothetical” Phosphoproteins Identified in Steady State and Disassembling Flagellaa

Spectral counts are in parentheses.

sample treatment and analysis was shown in Figure 1C. IMAC displays weak selectivity for monophosphopeptides over polyphosphopeptides. Thus, monophosphopeptides may be lost during enrichment, which results in bias identification toward polyphosphopeptides.38 Titanium dioxide (TiO2) chromatography is an alternative to IMAC. It has stronger selectivity for phosphopeptides and nonspecific binding of acidic unphosphorylated peptides can be easily minimized by the use of organic acids that act as excluders.39,40 However, polyphosphopeptides are difficult to elute. Furthermore, when a mixture of mono- and multiply phosphorylated peptides are analyzed together by mass spectrometry, the detection of polyphosphopeptides is suppressed. SIMC combines the strength of both IMAC and titanium dioxide chromatography. Mono- and polyphosphopeptides are sequentially eluted from IMAC. Monophosphopeptides are further enriched by removing nonphosphopeptides with TiO2 chromatography. Finally, eluted fractions of mono- and polyphosphopeptides are analyzed separately by tandem mass spectrometry. For phosphopeptide identification, two separate search algorithms including SEQUEST and OMSSA were used independently. The peptides identified from both searches were merged, generating a profile of all peptides identified. A merged database from Chlamydomonas genome v3.1 and v4 (http://genome.jgi-psf. org) and flagellar proteome33 was used for protein identification. Disassembling flagella were isolated twice. Phosphopeptides from each sample were collected and analyzed separately. The phosphopeptides and derived proteins from both measurements and analysis were merged, generating total phosphoproteins identified. For comparison with steady state flagella, flagella from steady growing cells were also isolated and analyzed as above. Phosphoproteins of Flagellar Membrane/Matrix

Our phosphoprotein analysis of flagellar membrane/matrix uncovered 224 phosphoproteins derived from around 1296 spectral counts of phosphopeptides (Table 1). All the phosphopeptides and corresponding proteins are listed in Supporting Information Table S1. These proteins were categorized into different groups based on their properties: Flagellar associated proteins (FAPs); flagellar motility related proteins; IFT proteins including IFT motor and IFT particle proteins; signaling proteins including protein kinases, phosphatases, and G proteins; ion channels or receptors; other known flagella-related proteins,;

193207

7 2

149909

2

294838 (Chlre4)

2

289945 (Chlre4) 166312

4 4 3 8

344556 (Chlre4)

1

5

193054

1

4

144918

2

5

205478 (Chlre4)

3

183037

2

4

a

The number of corresponding spectra counts is listed. Proteins with at least 1 fold change in total phosphopeptide spectra counts between control and disassembling flagella were included. C, control flagella; D, disassembling flagella. IDs are from Chlre3 assembly unless stated otherwise.

proteins of unknown function (unknown proteins, Table 2), and presumably nonflagellar proteins. All known flagellar phosphoproteins and FAPs are listed in Table 3 and Table 4, respectively. The major group of the identified phosphoproteins belongs to proteins with unkown functions, which comprises 49% (109 out of 224) phosphoproteins identified (Table S1). Future functional characterization of these proteins will be essential for full understanding of phosphorylation control of flagellar assembly, disassembly, and flagellar functions. Only 13 phosphoproteins seemed to be nonflagellar proteins, which include among others elongation factors, plastocyanin chloroplast precursor, tryptophan-tRNA ligase, and putative mitochondrial substrate carrier protein, indicating a high purity of the isolated flagella. Differential Protein Phosphorylation in Disassembling Flagella versus Control Flagella

We have identified 195 phosphoproteins from the membrane/ matrix fraction of shortening flagella (Table 1). The distribution of these proteins in different categories is displayed in Figure 2. To identify proteins that may be differentially phosphorylated during flagellar disassembly, we also analyzed phosphoproteins from flagella of steady state cells. A total of 135 phosphoproteins derived from around 497 spectral counts of phosphopeptides have been identified. In comparison with the phosphoproteins of shortening flagella, 29 unique phosphoproteins have been identified in control flagella, and 89 in shortening flagella (Table 1). 3833



TECHNICAL NOTE

Table 3. Major Functional Groups and Spectra Counts of Flagellar Phosphoproteins Present in Membrane/Matrix from Control or Disassembling Flagellaa functional group Flagellar Motility

Signaling Proteins

Intraflagellar Transport

Other Flagellar Proteins

a

protein name

ID (JGI Chlre3)

protein description

C

D

DHC7

347101

Dynein heavy chain

3

2

DHC8 ODA3b (ODA-DC1)

135994 189083

Dynein heavy chain Outer dynein arm 3

1 1

5 4

ODA4 (ODA-DHCb)

24252

Outer dynein arm 4

-

2

ODA9 (ODA-IC1)

129433

Outer dynein arm 9

-

1

ODA11 (ODA-DHCa)

130849

Outer dynein arm 11

12

18

PF6b

181848

Central pair protein

26

28

PF16

103782


2

4

CPC1/FBB7

24512


1

4

HYD3b KLP1

116240 186414

Hydin Kinesin like protein

7 7

11 9

PGM1a/PGM1b

161085

Phosphoglycerate kinase

2

3

MAPK5

131998

Protein kinase

3

1

PKGb

181974

Protein kinase

5

6

CNK4

132979

Protein kinase

3

5

CNK5

188335

Protein kinase

1

3

SKS-C

184895

Protein kinase

6

4

FAP295 FAP247

131695 102300

Cyclic nucleotide dependent kinase Protein Kinase

2 2

2 2

FAP262b

114241

Protein kinase

7

8

MAKb

(168935)

Protein kinase

14

22

CDPK

168407

Calcium dependent protein kinase

-

1

CALK

192550

Protein kinase

1

-

PP2A1

185509

Phosphatase

1

4

PKL1

101543

Protein Phosphatase 1 beta

1

1

TRP-2 RABB1

193017 148836

Ion channel Small G protein

-

2 2

Phototropin

183965

Photoreceptor

3

10

DHC1b

24009

Retrograde IFT motor

-

1

D1bLIC

130394

Retrograde IFT motor/light intermediate chain

-

1

IFT57

98642

IFT particle protein

1

-

IFT72/IFT74

136521

IFT particle protein

2

-

BUG2/FAP71

29687

Basal-body proteins with up-regulated gene 2

2

6

BUG15/FAP21 E2

191879 183594

Basal-body proteins with up-regulated gene 15 Ubiquitiin-conjugating enzyme E2

10 1

10 -

E3(UBC7)

190835

Ubiquitin ligase E3

-

1

MetE

154307

Vitamin B12 (cobalamin) independent methionine synthase

-

7

SAM (MetM)

182408

S-Adenosylmethionine synthetase

-

2

TUA1/TUA2

128523

Alpha tubulin

2

6

TUB1/TUB2

129868

Beta tubulin

3

12

C, control flagella; D, disassembling flagella. ID in parentheses is from jgi chlre2 database. b Indicates presence in analysis by Mittag and colleagues.29

The differential phosphorylation of known flagellar proteins is specified in Tables 3 and 4. Because of multisteps of sample processing during phosphopeptide enrichment and collection and the different abundance of the phosphoproteins, some phosphopeptides may have been differentially identified in one assay but not in other. Thus, experimental data is needed for further verification of differentially phosphorylated proteins. A total of 106 phosphoproteins were identified in both control and disassembling flagella. However, the phosphorylated residues and phosphopeptide numbers detected (Table S1) of the same protein under control and flagellar disassembly conditions may be different. This difference in phosphorylation extent and residues

may reflect specific regulatory and/or functional properties of these proteins during flagellar maintenance and disassembly. Mittag and colleagues have recently analyzed phosphoproteins from control flagella of Chlamydomonas and found 32 known flagellar phosphoproteins.29 Thirteen out of 32 phosphoproteins identified by Mittag and colleagues have been identified in our analysis (Tables 3 and 4). Though we have not detected all the phosphoproteins detected before, we have recognized a total of 62 known flagellar phosphoproteins in control flagella. The fact that we have identified more phosphoproteins may be due to several reasons. First, instead of using traditional IMAC, we have used SIMAC for phosphopeptide enrichment and collection, which was 3834



TECHNICAL NOTE

Table 4. Continued

Table 4. Flagella Associated Proteins (FAPs) in the Phosphoproteome of Control or Disassembling Flagella and Corresponding Spectra Countsa protein

ID (jgi chlre3)

protein b

C

D

FAP5

193372

1

3

FAP7

147217

8

18

FAP12

184308

19

30

FAP13

(159208)

3

2

FAP21 FAP26

191879 35808

10 3

10 1

FAP32

108954

11

13

FAP39b

118223

9

11

FAP43

193355

FAP48

194442

6

6

FAP55b

144113

2

5

FAP62

36026

20

18

FAP63 FAP69

193134 188246

8

1 13

FAP70

(153157)

3

3

FAP71

29687

2

6

FAP75

142494

8

12

FAP78

172392

1

1

FAP81

(158803)

FAP88

(159080)

3

3

FAP91 FAP92

(165613) 143281

1 1

1

FAP93b

190974

3

3

FAP96

196241

3

FAP101

141689

2

FAP111

188180

1

FAP116b

185392

5

FAP121

(163334)

7

8

FAP123 FAP125

188205 117642

1

5 2

FAP126

190291

FAP131

131208

5

6

FAP138

190644

8

21

FAP139

191309

3

4

FAP152b

185409

6

3

FAP157

180708

3

3

FAP165 FAP171

193605 147815

3

4 3

FAP176

175641

1

FAP178

53378

1

1

FAP181

152327

6

5

FAP185

(153893)

2

1

FAP190b

194620

21

17

ID (jgi chlre3)

FAP262 FAP268

144070 18398

FAP274

159889

FAP280

76570

FAP291

184735

FAP294

191852

FAP295

131695

C

D

7 2

8 2 1 1

9

11 1

2

2

C, control flagella; D, disassembling flagella. ID numbers in parentheses originate from the sequence collection of flagellar proteins33 and correspond to those of jgi chlre2. b Indicates presence in analysis by Mittag and colleagues.29 a

2

1

4 3

1

6

Figure 2. Grouping of identified phosphoproteins in shortening flagella. Pie chart showing the fraction of proteins categorized according to functions or other properties. The number of proteins in each category is listed in parentheses.

supposed to yield more phosphopeptides. Second, variations in sample treatment and processing from each analysis could also make a difference. Although we have identified more phosphoproteins, the fact that we have failed to identify 15 phosphoproteins identified by Mittag and colleagues indicates that independent analyses facilitate the definition of a whole flagellar phosphoproteome. Phosphorylation Control of Flagellar Disassembly through Ubiquitination

Ubiquitination is a post-translational modification process that involves covalent attachment of mono- or chained multiubiquitin molecules to lysine residues of target proteins. It is a three-step reactions sequentially catalyzed by ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3).41 Ubiquitin conjugation system is present in cilia and flagella. During flagellar disassembly, ubiquitinated proteins in the flagella are highly increased,16 indicating an active control of protein ubiquitination during flagellar disassembly. However, it is unknown how the activation of the ubiquitination system is controlled. We found that E2 (ID183594) is phosphorylated in control but not in disassembling flagella. In addition, E3 (UBC7, ID190835) was phosphorylated in shortening flagella with the following phosphopeptide: AVVPAEGAVGASDVQQQQAQHVADGstKPWK (only one single residue was phosphorylated) (Table 3, Table S1). This raises the possibility that the ubiquitin conjugating system may be regulated by protein phosphorylation. The activity of E3 ligase has been shown to be controlled by protein phosphorylation.42 E3 ligase in Chlamydomonas is likely regulated in similar manner.

FAP215

114150

FAP217b FAP222

146112 153689

1

5 2

FAP228b

143511

1

3

FAP232

(163277)

FAP247

102300

2

2

FAP254

36026

0

4

Phosphorylation Control of Flagellar Disassembly through Methylation

FAP256 FAP261

186225 192696

2

1 4

Protein methylation plays a role in controlling gene transcription, signaling and protein targeting.43 Protein methylation involves

1

3835



TECHNICAL NOTE

several steps of reactions. S-adenosylmethionine synthetase (SAM) catalyzes the formation from methionine of S-adenosylmethionine, which serves as methyl donor. The methyl group is then transferred to the target protein via methyltransferases. Methionine synthase (MetE), SAM, and methyltransferases have been identified in the Chlamydomonas flagella.33 Sloboda’s group showed that the amount of MetE increases in regenerating flagella and especially in shortening flagella. Furthermore, the quantity of methylated protein dramatically decrease during flagellar shortening.15 Since the increase in hydrophobicity and steric bulk by methylation could affect protein protein interactions, protein methylation may be involved in disassembling large protein complexes or associating disassembled components with IFT particles. We found that both MetE and SAM were specifically phosphorylated in shortening flagella (Table 3 and Table S1). In rat liver, phosphorylation of SAM at T342 dissociates inactive dimer and produces active monomer.44 It is thus likely that protein phosphorylation regulates the enzymatic activities for protein methylation during flagellar shortening.

phosphorylation of these and other proteins mentioned above in the central pair complex supports the view that protein phosphorylation of central pair proteins is a key regulator of flagellar motility.53 Flagellar beating also requires large amounts of ATP. The glycolytic pathway is present in Chlamydomonas flagella as well as mammalian sperm flagella.33 The key enzymes in the glycolytic pathway are under protein phosphorylation control.62 Interestingly, phosphoglycerate mutase (ID161085) was also found to be phosphorylated (Table 3).

Phosphorylation Control of Flagellar Motility

Phosphorylation Control of IFT

Cilia and flagella beat with various waveform and frequency. The inner dynein arms determine the size and shape of the waveform, whereas the outer dynein arms increase the beat frequency.7,45 The dynein activities are regulated by the central pair apparatus and radial spokes. It is proposed that the rotation of central pair transmits signals from central pair projections to radial spokes leading to selective activation of adjacent dynein arms.46 Protein phosphorylation plays a key role in this regulatory process. Protein kinase CKI, protein kinase A anchoring protein, as well as phosphatases PP1 and PP2A associate with flagellar axoneme and modulate dynein-driven microtubule sliding activities.7,47 The outer dynein arm is a large protein complex including heavy chains, light chains, and intermediate light chains and docks to the microtubule through docking complexes. The alpha dynein heavy chain (DHC) in Chlamydomonas,48 a 29 kDa light chain in Paramecium,49 and a 22 kDa light chain in sea urchin sperm flagella50 are phosphoproteins. We found phosphorylation of two outer arm DHCs: alpha (ODA11) and beta (ODA4), one docking complex (DC) protein DC1 (ODA3), and one intermediate chain IC1 (ODA9) (Table 3 and Table S1). The regulation of the activities of the outer dynein arms is not well studied. The identification of protein phosphorylation and phosphorylation sites in docking complex, outer dynein arm provides basis for further studying the assembly and function of outer dynein arms. Inner dynein arms can be phosphorylated, shown by in vivo pulse labeling with 32P, and so are several radial spoke proteins.51 The Inner dynein intermediate chain IC138 is a key regulator of flagellar motility and was found to be phosphorylated.52 Consistent with these studies, inner arm dynein heavy chain DHC7 and 8 were recognized to be phosphorylated (Table 3). Further experimental data are needed to demonstrate the roles, if any, of these phosphorylations in controlling flagellar motility. The central pair complex includes two single microtubules and associated projections. Disruption the structure by gene mutations impairs flagellar motility.53 Several proteins localized to the central pair complexes have been well studied. This includes PF6,54 PF16,55,56 CPC1,57 Hydin (HY3),58,59 and KLP1.60,61 Phosphorylation of Hydin, PF6, PF16, CPC1, and KLP1 was found in our current analysis with phosphorylation of KLP1 and PF6 being reported previously.29,60 Our identification of protein

Table 5. Phosphopeptides of IFT Proteins proteins

phosphopeptides

dhc1b

RLsELLAQVQAGLGTAVR

D1bLIC

sQGEGTVAGGLAEWR

IFT57

RLLQAQAALsDEEED

IFT74

MGTASQRPGtGQQAAAAAAAAR

IFT is required for flagellar assembly, maintenance, disassembly, and signaling. However, little is known about the regulation of the IFT machinery. In our phosphoproteome analysis, 4 IFT proteins were identified as phosphoproteins including IFT motor proteins (DHC1b, D1bLIC) and IFT particle proteins (IFT74 and IFT57) (Table 3). The recognized phosphopeptides were listed in Table 5. IFT 74, 57, and 25 are three IFT proteins that are identified or implicated to be phosphoproteins,63 65 though the phosphorylation sites and the responsible protein kinases have not been identified. Both IFT74 and 57 were identified in our analysis. Three properties of IFT protein may be regulated: IFT trafficking, IFT particles remodeling, and IFT cargo loading and unloading. During flagellar disassembly, IFT trafficking into the flagellum increases around 2- to 4-fold and the IFT cargo loading is suppressed at entry into the flagellum.18 During flagellar maintenance, IFT particles are remodeled.66,67 Further experimental data are needed to demonstrate whether protein phosphorylation is involved in these regulatory processes.

Protein Phosphorylation of Signaling Molecules

Sixteen signaling proteins were detected to be phosphorylated (Table 3). These include proteins kinase, phosphatase, ion channel, receptor, and small G proteins. Multiple protein kinases and phosphatases have been found in the flagellar proteome of Chlamydomonas.33 Chlamydomonas NIMA related kinases (CNKs) are a family of Nima-like protein kinases.68 Nima-like protein kinases have been shown to regulate flagellar assembly or disassembly in Chlamydomonas, Tetrahymena, and mammals.24,26,69 CNK4 and CNK5 were found in our phosphoproteomic analysis. Ion channels have been proposed to regulate flagellar length.70 Interestingly, we found TRP-2 was only phosphorylated in shortening flagella (Table 3, Table S1). In addition to protein kinases, phosphatases, and ion channels, small G protein RABB1 was found to be phosphorylated. RABB1 is present in the flagellar proteome.33 RABB1 has not been implicated in ciliary functions or ciliogenesis in other studies, though at least 7 small G proteins have been shown to be related to ciliogenesis.71 The human homologue of RABB1 is RAB2b, which is not detected in cilia by expressing an EGFP tagged form.72 However, the detection of RABB1 in the flagellar proteome as well as in our current study 3836


Journal of Proteome Research indicates that either RABB1 plays a specific role in Chlamydomonas flagella or its expression in mammalian cilia is low.

’ CONCLUSION We have used a SIMAC strategy to isolate and enrich phosphopeptides from disassembling and control flagella from Chlamydomonas, a model organism for ciliary studies. A total of 224 phosphoproteins have been identified from the flagella, including IFT proteins, signaling proteins, and flagellar motility-related proteins. This analysis reveals phosphoproteins present in steady state flagella as well as disassembling flagella, which provides basis for further mechanistic studies of flagellar assembly/disassembly and flagellar motility. Eighty-nine phosphoproteins were differentially identified in shortening flagella. This finding combined with previous work from others indicates that protein phosphorylation is a key regulatory event in flagellar disassembly.19 Because of multiple steps in sample processing prior to mass spectrometry analysis, less abundant phosphopeptides may have been differentially lost in our assays. Thus, additional experimental data are required to validate whether a protein is indeed differentially phosphorylated in steady state flagella or disassembling flagella. ’ ASSOCIATED CONTENT

bS

Supporting Information All the phosphopeptides and their spectral counts and derived proteins are listed Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Junmin Pan: e-mail, [email protected]; tel/fax, 86 10 62771864. Michael Hippler: email, [email protected]; tel., 49 251 83-24790; fax, 49 251 83-28371. Author Contributions #

These authors contributed equally.

’ ACKNOWLEDGMENT We are grateful to the DAAD fellowship (to J.P.). This work was supported by the German Science Foundation (FOR964, DFG) (to M.H.), National Natural Science Foundation of China (Grants 30830057, 30988004, and 30771084), National Basic Research Program of China [also called 973 program (Grant 2007CB914401) and SRFDP (to J.P.)]. ’ ABBREVIATIONS IMAC, immobilized metal affinity chromatography; SIMAC, sequential elution from IMAC ’ REFERENCES (1) Cavalier-Smith, T. Predation and eukaryote cell origins: a coevolutionary perspective. Int. J. Biochem. Cell Biol. 2009, 41, 307–22. (2) Mitchell, D. R. The evolution of eukaryotic cilia and flagella as motile and sensory organelles. Adv. Exp. Med. Biol. 2007, 607, 130–40. (3) Silflow, C. D.; Lefebvre, P. A. Assembly and motility of eukaryotic cilia and flagella. Lessons from Chlamydomonas reinhardtii. Plant Physiol. 2001, 127, 1500–7.

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Protein Phosphorylation Is a Key Event of Flagellar Disassembly

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