Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)
Summary
CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.
Abstract
Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.
Introduction
In most eukaryotes, spindle microtubules must attach to a single region of each chromosome, termed as centromere. The kinetochore is a complex of proteins that are located at the centromere. Studying the timing of centromere and kinetochore protein’s movements and the structure of kinetochores and centromeres is important for understanding chromosome instability (CIN) and cancer progression. The key questions are how the chromosomal location and function of a centromere (i.e., centromere identity) are determined and how they participate in accurate chromosome segregation. In most species, the presence of a special nucleosome containing a specific histone-like protein called CENP-A defines the centromere identity. Therefore, it is proposed that CENP-A is the non-DNA indicator (epigenetic mark) of centromere identity. It is important to elucidate the mechanism of how CENP-A defines the centromere identity in humans.
The Holliday junction recognition protein (HJURP) is the CENP-A-specific chaperon which deposits CENP-A in centromeric nucleosomes1,2,3. We have previously reported that the CUL4A-RBX1-COPS8 E3 ligase is required for CENP-A ubiquitylation on lysine 124 (K124) and centromere localization4. Also, our results showed that the centromere recruitment of newly synthesized CENP-A requires pre-existing ubiquitylated CENP-A5. Thus, a model was provided suggesting that CENP-A ubiquitylation is inherited through dimerization between cell divisions.
In contrast to our findings and those of Yu et al., negative results regarding the CENP-A and its centromeric localization were recently published6. The article claimed that CENP-A modifications on lysine 124 (K124) are dispensable for the establishment, maintenance, and long-term function of human centromeres, based on their negative results showing that the mutation of K124R did not affect CENP-A centromere localization neither cell viability6. However, there is enough room for debate in their results and conclusions, and we have already described what problem there could be in their previous publication7. Attention should be paid that they fused proteins with CENP-A, which have much larger molecular weights than the size of endogenous CENP-A: e.g., they fused ~30 kDa enhanced yellow fluorescent protein (EYFP) to ~16 kDa CENP-A and analyzed EYFP-CENP-A K124R fusion protein in their RPE-1 CENP-A-/F knockout system. K124 ubiquitin is not expected to bind directly to HJURP based on structural predictions4, however, addition of mono-ubiquitin is predicted to have an impact on protein conformation of CENP-A. The protein of CENP-A conformation can be changed by the presence of a large fusion protein, and this conformational change may mask the structural changes caused by the loss of ubiquitylation. We suggest that the fusion of large-sized protein induces ubiquitylation at a lysine other than K124 in EYFP-CENP-A K124R mutant and this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype. Evidence that ubiquitylation occurs at different lysine in the CENP-A K124R mutant protein with a large tag protein (EYFP) was reported in our previous publication8. It was found that EYFP tagging induces ubiquitination of another lysine site of EYFP-CENP-A K124R and that EYFP-CENP-A K124R mutant binds to HJURP. As a result, this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype, and both EYFP-CENP-A WT and K124R mutants showed centromere localization (we used and compared pBabe-EYFP-CENP-A WT and K124R mutant, together with pBabe-EYFP control.). The results demonstrated that Flag-tagged or untagged CENP-A K124R mutants are lethal but can be rescued by a monoubiquitin fusion, suggesting that CENP-A ubiquitylation is indispensable to cell viability.
In recent years, many studies have developed different assays to identify posttranslational modifications (PTMs) of CENP-A protein and other centromere-kinetochore proteins both in vivo and in vitro9,10,11. Analogous to the PTMs of histone proteins that are a major mechanism regulating the function of chromatin, PTMs of centromeric chromatin components are also involved in an essential mechanism to regulate the overall structure and function of centromeres. The majority of CENP-A PTM sites are specific to CENP-A-containing nucleosomes, although a few of them are conserved in histone H3, suggesting that modification of these residues contribute to the centromere-specific function. PTMs of CENP-A including phosphorylation, acetylation, methylation, and ubiquitylation were previously reported9, suggesting that CENP-A is subjected to a variety of PTMs and their combinatorial arrays on its amino terminus and C-terminus histone-fold domain. The importance of CENP-A modifications in multiple functions was revealed by many groups including ours. These functions involve CENP-A deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network)9. However, limited studies and findings of CENP-A PTMs are preformed where comparisons are made with one of canonical histones that directly or indirectly regulate their function. Technical reports focusing on the methodology to identify these CENP-A PTMs are also limited.
Because CENP-A ubiquitylation is required for CENP-A deposition at the centromere12, inherited through dimerization between cell division5, and indispensable to cell viability8, the method to identify CENP-A ubiquitylation would be essential in future to study the functional activity, positioning, and structure of the centromere. Therefore, here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that the EYFP tagging induces ubiquitylation at a different lysine in the CENP-A K124R mutant protein8. Protocols of other control assays and analyses (immunofluorescence analysis, colony outgrowth assay, and in vivo ubiquitylation assay) are also presented to discuss the outcome of major mass spectrometry analysis properly.
Protocol
Representative Results
Discussion
Here we described methods of mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that the EYFP tagging induces ubiquitylation at a different lysine in the CENP-A K124R mutant protein8. In our results, we successfully identified ubiquitylation on lysine 306 (K306) in EYFP-CENP-A K124R, that is corresponding to lysine 56 (K56) in CENP-A through mass spectrometry analysis. The mass spectrometry analysis described here is a mimic method as we previously identif…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Chao-Jun Li at the Model Animal Research Center, Nanjing University for mass spectrometry analysis. We thank Yanmini Dalal, Tatsuo Fukagawa, and current researchers at the Model Animal Research Center, Nanjing University and Greehey Children’s Cancer Research Institute for their helpful discussion, experimental guidance, and reagents. We thank Don W. Cleveland, Daniele Fachinetti, Yanmini Dalal, Minh Bui, Gustavo W. Leone, John Thompson, Lawrence S. Kirschner, Amruta Ashtekar, Ben E. Black, Glennis A. Logsdon, Kenji Tago, and Dawn S. Chandler for their generous gifts of reagents. Y.N. was supported by Jiangsu Province ‘‘Double- First-Class’’ Construction Fund, Jiangsu Province Natural Science Fund (SBK2019021248), Jiangsu Province 16th Six Big Talent Peaks Fund (TD-SWYY-001), Jiangsu Province “Foreign Expert Hundred Talents Program” Fund (SBK2019010048), and National Natural Science Foundation in China (31970665). This study was partly supported by NCI grant R21 CA205659.
Materials
| Equpiments/Tools | |||
| 0.5 ml protein low binding tubes | Eppendorf | 022431064 | For mass spectrometry analysis |
| 10cm cell culture dish | BIOFIL/JET, China | 700224 | 10 cm tissue culture dish (Yohei lab, PN63) |
| 6 Well Cell Culture Cluster | Fisher/Corning Incorporated | 07-200-83 | 6-well culture plate |
| CentriVap | LABCONCO | – | Benchtop vacuum concentrator for vaccum dry peptides for mass spectrometry analysis |
| ChromXP C18CL, 120A, 15 cm x 75 μm | Eksigent Technologies | 805-00120 | Liquid chromatography (RPLC) column for mass spectrometry analysis |
| HCX PL APO 100x oil immersion lens | Leica | LEICA HCX PL APO NA 1.40 OIL PHE | 100X Oil immersion lens |
| HCX PL APO 63x oil immersion lens | Leica | LEICA HCX PL APO NA 1.40 OIL PH 3 CS | 63X Oil immersion lens |
| Immobilon-FL PVDF Transfer Membrane | EMD Millipore | IPVH00010 | For western blot |
| Leica DM IRE2 motorized fluorescence microscope | Leica | – | motorized fluorescence microscope |
| Leica EL6000 compact light source | Leica | External light source for fluorescent excitation | |
| Micro Cover glass (22 mm x 22 mm) | Surgipath | 105 | Cover glass (22 mm x 22 mm) |
| Model V16-2 polyacrylamide gel electrophoresis apparatus | Apogee Electrophoresis/CORE Life Sciences | 31071010 | Gel electrophoresis apparatus I to apply bigger SDS-PAGE gel |
| nanoLC.2D | Eksigent Technologies | – | liquid chromatography system for mass spectrometry analysis |
| NuPAGE 4%-12% Bis-Tris Protein Gels | Thermo Fisher | NP0335BOX | The commercially available 4%-12% Bis-Tris protein gels for mass spectrometry analysis |
| Olympus FLUOVIEW FV3000 confocal laser scanning microscope | Olympus | – | Confocal laser scanning microscope (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519) |
| ORCA-R2 Degital CCD camera | Hamamatsu | C10600-10B | CCD camera |
| PAP Pen | Binding Site | AD100.1 | For a water repellant barrier in immunofluorescent staining |
| TISSUE CULTURE DISHES 10CM | VWR | 25382-166 | 10 cm tissue culture dish |
| Vertical electrophoresis for gel running (big size) | Junyi, China | JY-SCZ6+ | Gel electrophoresis apparatus II to apply bigger SDS-PAGE gel (Yohei lab, PE23) |
| VWR Micro Slides, Frosted | VWR International | 48312-013 | Micro slides |
| Primary antibodies | |||
| Anti-CENP-A antibody | Stressgen/Enzo Life Sciences | KAM-CC006 | Mouse monoclonal antibody |
| Anti-CENP-B antibody | Novus Biologicals | H00001059-B01P | Mouse monoclonal antibody |
| anti-GAPDH | ABCAM | ab37168 | Rabbit polyclonal antibody |
| anti-GAPDH | Invitrogen | PA1987 | Rabbit polyclonal antibody |
| anti-GFP antibody | ANTI #76 (Homemade antibody) | Rabbit polyclonal antibody | |
| anti-HA (3F10) | Roche | 11815016001 | Rat monoclonal antibody |
| anti-HJURP | Proteintech Group | 15283–1-AP | Rabbit polyclonal antibody |
| anti-Ubiquitin | Bethyl Laboratories | A300-317A-1 | Rabbit polyclonal antibody |
| Reagents | |||
| Bio-Rad Protein Assay | Bio-Rad | 500-0006 | Commercial protein assay reagent I for measurement of protein concentration (compatible with 0.1% SDS) |
| Branson SONIFIER 450 | Sonicator | ||
| Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm | VWR Scientific Products Inc. | 33995-325 | Disruptor horn for sonication |
| Branson Ultrasonics sonicator Microtip Tapered 6.5 mm | VWR Scientific Products Inc. | 33996-185 | Microtip for sonication |
| Buffer A1 | – | – | 20 mM Tris-HCl, pH 7.4; 50 mM NaCl; 0.5% Nonidet P-40; 0.5% deoxycholate; 0.5% SDS; 1 mM EDTA; complete EDTA-free protease inhibitor reagent |
| Complete EDTA-free protease inhibitor cocktail | Roche | 11-873-580-001 | Complete EDTA-free protease inhibitor reagent for buffer A1 |
| Coomassie brilliant blue R-250 | BBI Life Sciences | CAS 6104-59-2 | Coomassie blue solution for mass spectrometry analysis |
| Crystal violet solution (2.3% crystal violet, 0.1% ammonium oxylate, 20% ethanol) | SigmaI-Aldrich | HT90132-1L | For colony staining |
| DAPI | SIGMA-SLDRICH | D9542 | For nuclear staining |
| DMEM: F12 Medium | ATCC | 30-2006 | DMEM: F12 Medium |
| Fetal Bovine Serum, certified, heat inactivated, US origin | Life Technologies/Gibco | 10082 | FBS (fetal bovine serum) |
| High-glucose DMEM (Dulbecco’s modified Eagle’s medium) | Life Technologies/BioWhittaker | 12-604 | high-glucose DMEM |
| Lipofectamin 3000 | Life Technologies/Invitrogen | L3000 | Transfection reagent I for chemical transfection |
| Lipofectamin 3000, P3000 solution | Life Technologies/Invitrogen | L3000 | Transfection reagent II for chemical transfection |
| Methanol | Fisher | A412-4 | Fixation reagant |
| Non fat powdered milk (approved substitution for carnation powdered milk) | Fisher Scientific | NC9255871 (Reorder No. 190915; Lot# 90629) | Non-fat skim milk |
| Opti-MEM I | Life Technologies/Invitrogen | 31985 | Reduced serum media |
| p-phenylenediamine | SIGMA-SLDRICH | P6001 | For mounting medium |
| Penicillin, Streptomycin; Liquid | Fisher/Gibco | 15-140 | Penicillin-streptomycin |
| Poly-L-Lysine SOLUTION | SIGMA-SLDRICH | P 8920 | Poly-L-Lysine, 0.1% w/v, in water |
| Polyethyleneimine [PEI]; 1.0 mg/ml | Polysciences | 23966–2 | Transfection reagent III for chemical transfection |
| Protein A sepharose CL-4B beads | GE Healthcare/Amersham | 17-0963-03 | Protein A sepharose CL-4B beads for in vivo ubiquitylation assays using pQCXIP-EYFP-CENP-A |
| Restore Western Blot Stripping Buffer | Thermo Scientific | PI21059 | Western Blot Stripping Buffer I |
| Sequencing grade trypsin | Promega | V5111 | For mass spectrometry analysis |
| SuperSignal West Femto Maximum Sensitivity Substrate | Thermo | 34095 | Ultra-sensitive enhanced chemiluminescent (ECL) substrate |
| UltraPure Distilled Water | Life Technologies/Invitrogen/Gibco | 10977 | Sterile tissue culture grade water |
| Western Blot Stripping Buffer II ((50 mM Tris-HCl, pH 6.85; 2% SDS; 50 mM DTT; 100 mM 2-Mercaptoethanol) | – | – | Western Blot Stripping Buffer II |
| Secondary antibodies | |||
| Alexa Fluor 488 Goat Anti-Rabbit IgG | Life Technologies/Invitrogen | A11008 | fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody) |
| Alexa Fluor 594 Goat Anti-Mouse IgG | Life Technologies/Invitrogen | A11005 | fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody) |
| Softwares | |||
| Acquisition FV31S-SW software | Olympus | – | Sofware C1 (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519) |
| Analysis FV31S-DT software | Olympus | – | Sofware C2 (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519) |
| cellSens Dimension software Ver. 1. 18 | Olympus | – | Sofware C3 (https://www.olympus-lifescience.com.cn/en/ software/cellsens/) |
| Image Studio Analysis Software Ver 4.0 | LI-COR Biosciences | – | Software D |
| Molecular Imager Versadoc MP4000 System | Bio-Rad | – | Chemiluminescence imager for immunoblot detection |
| Odyssey CLx Infrared imaging System | LI-COR Biosciences | – | Infrared imaging system for immunoblot detection |
| OpenCFU saftware | – | – | For colony counting (http://opencfu.sourceforge.net/) |
| Openlab version 5.5.2. Scientific Imaging Software | Improvision/PerkinElmer | – | Software A |
| ProteinPilot Software version 4.5 | AB SCIEX | – | Software F for mass spectrometry analysis |
| Quantity One 1-D analysis software | Bio-Rad | – | Software E |
| TripleTOF 5600+ System | AB SCIEX | – | Mass spectrometry instrument |
| Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition) | PerkinElmer | – | Software B1 |
| Volocity version 6.3 3D Image Analysis Software (Volocity Quantification) | PerkinElmer | – | Software B2 |
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