Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Summary

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.

Abstract

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.

Introduction

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 eukaryotes^1,2,3,4,5, and phosphorylates an estimated 8-13% of the proteome^6,....

Protocol

1. Prediction of Cdk1-specific Phosphorylation Sites from the Protein Sequence

1. Before beginning the kinase assay, analyze the protein sequence for predicted Cdk1-specific phosphorylation sites and search the literature for experimentally established phosphorylation sites with unknown kinase specificity. Use the following tools, databases, and references that are summarized.
   1. Use the iGPS 3.0 software^36,37 (http://gps.biocuckoo.org/online_fu.......

Discussion

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.......

Materials

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