Name: identification of cyclin-dependent kinase 1 specific phosphorylation sites by an in vitro kinase assay
Uploaded: 2018-05-03T15:00:00
Description: Watch this Scientific Journal Video about Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay at JoVE.com

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.

1. Prediction of Cdk1-specific Phosphorylation Sites from the Protein Sequence
<ol>
	<li>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.
	<ol>
		<li>Use the iGPS 3.0 software36,37 (http://gps.biocuckoo.org/online_fu...

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...

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 proteome6,7.
While recent proteomic studies have identified many phosphorylation sites in proteins, in most cases, the kinase responsible for these modifications is unknown. The number of known Cdk1 targets, particularly in human cells is low7. The identification of Cdk1-specific phosphorylation sites is important, as it enables mechanistic studies that establish how Cdk1 controls the cell cycle. Cell cycle regulation is important for faithful chromosome segregation and cell division, and a myriad of cellular processes need to occur to support this important physiological function. This includes halting transcription and translation prior to the onset of mitosis, as well as a dramatic reorganization in cellular structure and organization, such as disassembly of the nuclear envelope, chromosome condensation, and mitotic spindle assembly. Deregulation and errors in these processes cause cancer, birth defects, or mitotic cell death. Specific inhibitors of Cdk1 such as RO-3306 were developed8, which provide powerful tools for functional studies, and some of these inhibitors are currently in clinical trials for cancer treatment (see9 for review).
Here, we describe an in vitro kinase assay that allows the identification of Cdk1-specific phosphorylation sites. In this assay, commercially available human Cdk1/cyclin B is used to phosphorylate a purified target protein in vitro. Phosphorylation of a substrate increases its mass and adds two negative charges; therefore, successful phosphorylation is confirmed by an upward shift of the protein gel band on SDS-PAGE. Cdk1-specific phosphorylation sites are subsequently identified by mass spectrometry analysis of the in vitro phosphorylated protein. To aid with experimental design, we also review computational tools and references for the prediction of Cdk1-specific phosphorylation sites from the protein sequence. Furthermore, we also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay. Finally, the identified phosphorylation sites must be verified by functional studies, and a simple binding assay is described here for that purpose. Combined, this is a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle7,10,11. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results. However, functional verification of the obtained phosphorylation sites in vitro is recommended, as cells have additional regulatory mechanisms in place, such as posttranslational modifications, interaction partners, or cellular localization that may render phosphorylation sites accessible or inaccessible for recognition by Cdk1.
Cdk1 recognizes a consensus phosphorylation site that consists of (Ser/Thr-Pro-X-Lys/Arg), where X is any residue and a serine or threonine is the site of phosphorylation. Especially important for recognition is the presence of the proline in the +1 position. In addition, basic residues are preferred in the +2 or +3 positions, with most Cdk1-specific phosphorylation sites containing a Lys or Arg at the +3 position6,12.
Activation of Cdk1 is tightly regulated and leads to the onset of mitosis1,2,3,4,5. The activity of cyclin-dependent kinases in general depends on their association with distinct cyclins (cyclin A, B, C, D, and E in humans), which are expressed at oscillating levels throughout the cell cycle13. Cdk1 expression is constant across the cell cycle and the regulation of its activity relies on its association with the regulatory subunits cyclin A and cyclin B5,13,14,15, as well as post-translational modifications. Formation of the Cdk1/cyclin B complex is required for the kinase activation5,14,15,16,17,18. In the G2 phase, cyclin B is translated in the cytosol and imported into the nucleus where it binds to Cdk15,14,15,16,17,18; however, Cdk1/cyclin B is held inactivated by phosphorylation at residues Thr14 and Tyr15 by the human Cdk1-inhibitory kinases Myt1 (membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase) and Wee1, respectively19,20,21. In the late G2 phase, dephosphorylation of Thr14 and Tyr15 by cell division cycle 25 phosphatase (cdc25) activates the kinase activity of the Cdk1/cyclin B complex and triggers the initiation of mitosis12,14,18,20,22,23. Phosphorylation of Thr161 is also required for Cdk1/cyclin B activation and is mediated by Cdk7, the Cdk-activating kinase (CAK)18. Degradation of cyclin B in anaphase inactivates Cdk1, allowing exit from mitosis24,25. Activation of Cdk1/cyclin B is therefore a complicated process. The protocol presented here is performed with commercially available Cdk1/cyclin B. During recombinant expression of this complex in insect cells, it is activated in vivo by endogenous kinases14,20 and remains active in the purified state. The resulting active, recombinant human Cdk1/cyclin B is suitable for in vitro kinase assays.
Here, we describe a protocol for the identification of Cdk1-specific phosphorylation sites in the human centromere protein F (CENP-F)10. CENP-F is a kinetochore protein that resides in the nucleus during most of interphase (G1 and S-phase) and is exported to the cytosol in the G2 phase26,27,28 in a Cdk1-dependent manner10,11. Nuclear localization is conferred by a bipartite cNLS26. cNLSs are recognized by the nuclear transport factor karyopherin &#945;, which facilitates, together with karyopherin &#946; and RanGDP, the import of cNLS-cargo into the nucleus29. Nuclear export in the G2 phase is facilitated via an unknown export pathway10. Once CENP-F resides in the cytosol, it is recruited to the nuclear envelope and in turn recruits the motor protein complex dynein30,31. This pathway is important to position the nucleus respective to the centrosome during initial stages of mitotic spindle assembly in a dynein-dependent manner, which is important for the correct timing of mitotic entry and for a fundamental process in brain development30,31,32. Starting in the G2 phase, CENP-F is also assembled into the kinetochore where it has important roles for faithful chromosome segregation27,28,33,34,35. A key regulatory step of these pathways is the nuclear export of CENP-F in the G2 phase, which is dependent on Cdk110,11. We describe here a protocol for the identification of Cdk1-specific phosphorylation sites in the cNLS of CENP-F. Phosphomimetic mutations of these sites slow down nuclear import of CENP-F, suggesting that Cdk1/cyclin B directly regulates cellular localization of CENP-F by phosphorylation of its cNLS10.
Overall, this in vitro kinase assay allows the identification of specific substrates for the kinase Cdk1. Purified target proteins are phosphorylated in vitro by the commercially available Cdk1/cyclin B complex and the phosphorylation sites are subsequently identified by mass spectrometry. The identification of Cdk1-specific phosphorylation sites supports mechanistic studies that reveal how Cdk1 controls the cell cycle.

<table>
	<tbody>
		<tr>
			<td>2800 ml baffled Fernbach flask</td>
			<td>Corning</td>
			<td>44232XL</td>
			<td></td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Gold Biotechnology</td>
			<td>A-301-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Fisher Scientfiic</td>
			<td>BP413-25</td>
			<td></td>
		</tr>
		<tr>
			<td>benzamidine hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>B6506-25</td>
			<td></td>
		</tr>
		<tr>
			<td>bottletop filter</td>
			<td>Corning</td>
			<td>431161</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B recombinant, human 20,000 U/mL</td>
			<td>New England Biolabs</td>
			<td>P6020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B (alternate source)</td>
			<td>EMD Millipore</td>
			<td>14-450</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B (alternate source)</td>
			<td>Invitrogen</td>
			<td>PV3292</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B + 10x PK buffer</td>
			<td>New England Biolabs</td>
			<td>P6020</td>
			<td></td>
		</tr>
		<tr>
			<td>CENP-F (residues 2987 &#8211; 3065) pGEX6P1 plasmid</td>
			<td>Available upon request.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge: Heraeus Multifuge X3R, cooled, with TX-1000 swing-out rotor</td>
			<td>Thermo Scientific</td>
			<td>10033-778</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifugal filter units: Amicon Ultra-15 centrifugal filter units, 3 kDa cutoff, Ultracel-PL membranes</td>
			<td>EMD Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>chlorampenicol</td>
			<td>Gold Biotechnology</td>
			<td>C-105-100</td>
			<td></td>
		</tr>
		<tr>
			<td>D/L methionine</td>
			<td>Agros Organics / Fisher</td>
			<td>125650010</td>
			<td></td>
		</tr>
		<tr>
			<td>desalting pipet tips: Zip tips</td>
			<td>Millipore Sigma</td>
			<td>ZTC18S008</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable chromatography columns, Econo-Pac 1.5 x 12 cm</td>
			<td>Biorad</td>
			<td>7321010</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Gold Biotechnology</td>
			<td>DTT50</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli Rosetta 2(DE3)pLysS strain</td>
			<td>EMD Millipore</td>
			<td>71403</td>
			<td></td>
		</tr>
		<tr>
			<td>electrospray ionization Fourier transform ion</td>
			<td>Bruker Amazon</td>
			<td>Apex III</td>
			<td></td>
		</tr>
		<tr>
			<td>cyclotron resonance mass spectrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electrospray ionization ion trap mass spectrometer</td>
			<td>Bruker Amazon</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>fixed angle rotor: Fiberlite F15-8x-50cy</td>
			<td>Thermo Scientific</td>
			<td>97040-276</td>
			<td></td>
		</tr>
		<tr>
			<td>FPLC system: &#196;kta Pure FPLC</td>
			<td>GE Healthcare</td>
			<td>29032697</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel filtration column: Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>glutathione agarose</td>
			<td>Pierce</td>
			<td>16101</td>
			<td></td>
		</tr>
		<tr>
			<td>glutathione, reduced</td>
			<td>Millipore Sigma</td>
			<td>G4251-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>incubation shaker: multitron shaker</td>
			<td>Infors</td>
			<td>I10102</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Gold Biotechnology</td>
			<td>I2481C50</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Gold Biotechnology</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>karyopherin &#945; pet-28a pres plasmid</td>
			<td>Available upon request.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani medium</td>
			<td>Fisher Scientfiic</td>
			<td>BP1426-2</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge 5418R, refrigerated</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (0.5 ml)</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (1.5 ml)</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel affinity gel: His-Select Nickel affinity gel</td>
			<td>Millipore Sigma</td>
			<td>P6611-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-6P-1 plasmid</td>
			<td>Millipore Sigma</td>
			<td>GE28-9546-48</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Gold Biotechnology</td>
			<td>P470-10</td>
			<td></td>
		</tr>
		<tr>
			<td>PS protease: PreScission protease</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Phos-tag acrylamide</td>
			<td>Wako Pure Chem. Ind.</td>
			<td>304-93521</td>
			<td></td>
		</tr>
		<tr>
			<td>reduced gluthathione</td>
			<td>Millipore Sigma</td>
			<td>G4251-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>roundbottom centrifuge tubes (Oakridge tubes)</td>
			<td>Fisher Scientfiic</td>
			<td>055291D</td>
			<td></td>
		</tr>
		<tr>
			<td>site-directed mutagenesis kit: QuikChange Lightning</td>
			<td>Agilent</td>
			<td>210518</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-Directed Mutagenesis Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sonifier: Branson S-250D sonifier</td>
			<td>Branson</td>
			<td>15 338 125</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra/Por 1RC dialysis membrane (6-8 kDa cutoff)</td>
			<td>Spectrum Labs</td>
			<td>08 670B</td>
			<td></td>
		</tr>
		<tr>
			<td>swing out rotor TX-1000</td>
			<td>Thermo Scientific</td>
			<td>10033-778</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of cyclin-dependent kinase 1 specific phosphorylation sites by an in vitro kinase assay

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 &#945;. 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.

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...

Watch this Scientific Journal Video about Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay at JoVE.com

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

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.

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

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; ...

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