Name: identification of cyclin-dependent kinase 1 specific phosphorylation sites by an in vitro kinase assay
Uploaded: 2018-05-03T15:00:00.000+00:00
Duration: 12 min 26 s
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.

The authors have nothing to disclose.

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 &amp;ndash; 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>&lt;em&gt;E. coli&lt;/em&gt; 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: &amp;Auml;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 &amp;beta;-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 &amp;alpha; 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; ...

identification-cyclin-dependent-kinase-1-specific-phosphorylation

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

18.5K Views.  State University of New York at Binghamton. 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.

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

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Biology

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

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

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Identification of Post-translational Modifications of Plant Protein Complexes

Identification of Kinase-substrate Pairs Using High Throughput Screening

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1