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* These authors contributed equally
Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.
Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.
Kinases are enzymes that transfer phosphate groups from ATP onto substrates and regulate many cellular processes. This phosphorylation is reversible, fast, adds two negative charges, and stores free energy, and is one of the most common posttranslational modifications used by cells. Cdk1, which is also known as cell division cycle protein 2 homolog (cdc2) is a master controller for the cell cycle in all eukaryotes1,2,3,4,5, and phosphorylates an estimated 8-13% of the proteome6,....
1. Prediction of Cdk1-specific Phosphorylation Sites from the Protein Sequence
We have recently used an in vitro kinase assay (Figure 1) to identify Cdk1-specific phosphorylation sites in a CENP-F fragment that contained a cNLS10. This signal confers nuclear localization of CENP-F during most of interphase. In the G2 phase, CENP-F is exported from the nucleus to the cytosol in a Cdk1-dependent manner. To obtain mechanistic insights on how Cdk1 regulates cellular localization of CENP-F, we analyzed the se.......
Our in vitro kinase assay is a very powerful method to identify molecular targets for the kinase Cdk1, which is a master controller of the cell cycle and regulates many important cellular processes. The method determines if a purified protein is a substrate for Cdk1 and allows identification of specific phosphorylation sites. This facilitates mechanistic studies for regulation of cellular processes by phosphorylation through Cdk1.
The most critical factor for successful identification.......
We thank Dr. David King, Howard Hughes Medical Institute, University of California at Berkeley for mass spectrometry analysis and helpful comments. We thank Dr. Xuelian Zhu, Shanghai, Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China for providing a full-length CENP-F construct. Finally, we thank Dr. Susan Bane, Dr. Brian Callahan and Dr. Christof Grewer at Binghamton University for access to equipment. This research was funded by the Research Foundation for the State University of New York and the Department of Chemistry, State University of New York at Binghamton.
....Name | Company | Catalog Number | Comments |
2800 ml baffled Fernbach flask | Corning | 44232XL | |
ampicillin | Gold Biotechnology | A-301-25 | |
ATP | Fisher Scientfiic | BP413-25 | |
benzamidine hydrochloride | Millipore Sigma | B6506-25 | |
bottletop filter | Corning | 431161 | |
Cdk1/cyclin B recombinant, human 20,000 U/mL | New England Biolabs | P6020 | |
Cdk1/cyclin B (alternate source) | EMD Millipore | 14-450 | |
Cdk1/cyclin B (alternate source) | Invitrogen | PV3292 | |
Cdk1/cyclin B + 10x PK buffer | New England Biolabs | P6020 | |
CENP-F (residues 2987 – 3065) pGEX6P1 plasmid | Available upon request. | ||
centrifuge: Heraeus Multifuge X3R, cooled, with TX-1000 swing-out rotor | Thermo Scientific | 10033-778 | |
centrifugal filter units: Amicon Ultra-15 centrifugal filter units, 3 kDa cutoff, Ultracel-PL membranes | EMD Millipore | UFC900324 | |
chlorampenicol | Gold Biotechnology | C-105-100 | |
D/L methionine | Agros Organics / Fisher | 125650010 | |
desalting pipet tips: Zip tips | Millipore Sigma | ZTC18S008 | |
disposable chromatography columns, Econo-Pac 1.5 x 12 cm | Biorad | 7321010 | |
dithiothreitol | Gold Biotechnology | DTT50 | |
E. coli Rosetta 2(DE3)pLysS strain | EMD Millipore | 71403 | |
electrospray ionization Fourier transform ion | Bruker Amazon | Apex III | |
cyclotron resonance mass spectrometer | |||
electrospray ionization ion trap mass spectrometer | Bruker Amazon | custom | |
fixed angle rotor: Fiberlite F15-8x-50cy | Thermo Scientific | 97040-276 | |
FPLC system: Äkta Pure FPLC | GE Healthcare | 29032697 | |
Gel filtration column: Superdex 200 Increase 10/300 GL | GE Healthcare | 28990944 | |
glutathione agarose | Pierce | 16101 | |
glutathione, reduced | Millipore Sigma | G4251-50g | |
incubation shaker: multitron shaker | Infors | I10102 | |
isopropyl β-D-1-thiogalactopyranoside | Gold Biotechnology | I2481C50 | |
kanamycin | Gold Biotechnology | K-120-25 | |
karyopherin α pet-28a pres plasmid | Available upon request. | ||
Luria Bertani medium | Fisher Scientfiic | BP1426-2 | |
microcentrifuge 5418R, refrigerated | Eppendorf | 5401000013 | |
microtubes (0.5 ml) | Eppendorf | 30121023 | |
microtubes (1.5 ml) | Eppendorf | 30120086 | |
Nickel affinity gel: His-Select Nickel affinity gel | Millipore Sigma | P6611-100ml | |
pGEX-6P-1 plasmid | Millipore Sigma | GE28-9546-48 | |
phenylmethylsulfonyl fluoride | Gold Biotechnology | P470-10 | |
PS protease: PreScission protease | GE Healthcare | 27084301 | |
Phos-tag acrylamide | Wako Pure Chem. Ind. | 304-93521 | |
reduced gluthathione | Millipore Sigma | G4251-50g | |
roundbottom centrifuge tubes (Oakridge tubes) | Fisher Scientfiic | 055291D | |
site-directed mutagenesis kit: QuikChange Lightning | Agilent | 210518 | |
Site-Directed Mutagenesis Kit | |||
sonifier: Branson S-250D sonifier | Branson | 15 338 125 | |
Spectra/Por 1RC dialysis membrane (6-8 kDa cutoff) | Spectrum Labs | 08 670B | |
swing out rotor TX-1000 | Thermo Scientific | 10033-778 |
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