Wir danken Dr. David King, Howard Hughes Medical Institute, University of California in Berkeley für Massenspektrometrie Analyse und hilfreichen Kommentare. Wir danken Dr. Xuelian Zhu Shanghai Institutes for Biological Sciences, chinesische Akademie von Wissenschaften, Shanghai, China für die Bereitstellung eines Full-Length CENP-F-Konstrukt. Zu guter Letzt danken wir Dr. Susan Bane, Dr. Brian Callahan und Dr. Christof Grewer an der Binghamton University für den Zugriff auf Geräte. Diese Forschung wurde von der Forschungsgemeinschaft für die State University of New York und der Fakultät für Chemie, State University of New York at Binghamton finanziert.

Die Autoren haben nichts preisgeben.

Unsere in-vitro- Kinase Assay ist eine sehr leistungsfähige Methode zur Identifizierung von molekularer Zielstrukturen für die Kinase Cdk1, die eine übergeordnete Steuerung des Zellzyklus und reguliert viele wichtige zelluläre Prozesse. Die Methode bestimmt, ob ein gereinigtes Protein ein Substrat für Cdk1 ist und ermöglicht die Identifizierung von spezifischen Phosphorylierung Websites. Dies erleichtert die mechanistische Studien zur Regulation zellulärer Prozesse durch Phosphorylierung durch Cdk1.
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Kinasen sind Enzyme, die Übertragung von Phosphatgruppen von ATP auf Substraten und regulieren viele zelluläre Prozesse. Diese Phosphorylierung ist reversibel, schnell, fügt zwei negative Ladungen und freie Energie speichert und gehört zu den häufigsten posttranslationale Modifikationen von Zellen verwendet. Cdk1, die auch bekannt als Zellteilung Zyklus Protein 2 Homolog (cdc2) ist ein master-Controller für den Zellzyklus in allen Eukaryoten1,2,3,4,5, und phosphorylates ein Geschätzte 8 bis 13 % des Proteoms6,7.
Während Proteomik-Studien haben viele Phosphorylierung Websites in den Proteinen, in den meisten Fällen identifiziert ist die Kinase verantwortlich für diese Änderungen unbekannt. Die Anzahl der bekannten Cdk1 Zielen, vor allem in menschlichen Zellen ist niedrig7. Die Identifizierung von Cdk1-spezifische Phosphorylierung Websites ist wichtig, da es mechanistische Studien, die festlegen ermöglicht, wie Cdk1 Zellzyklus steuert. Regulierung des Zellzyklus ist wichtig für Treue Chromosom Segregation und Zellteilung, und eine Vielzahl von zellulären Prozessen auftreten, um diese wichtige physiologische Funktion unterstützen müssen. Dazu gehören aufzuhalten, Transkription und Translation vor dem Beginn der Mitose sowie eine dramatische Reorganisation in zellulare Struktur und Organisation, z. B. Demontage der Kernhülle, Chromosom Kondensation und Montage der mitotischen Spindel. Deregulierung und Fehler in diesen Prozessen verursachen Krebs, Geburtsschäden oder mitotische Zelltod. Spezifische Inhibitoren des Cdk1 wie RO-3306 waren entwickelten8, die bieten leistungsstarke Tools für funktionelle Studien, und einige dieser Inhibitoren sind derzeit in klinischen Studien zur Behandlung von Krebs (siehe9 zur Überprüfung).
Hier beschreiben wir einen in-vitro- Kinase Assay, der die Identifizierung von Cdk1-spezifische Phosphorylierung Websites ermöglicht. In diesem Test wird kommerziell verfügbare menschliche Cdk1/cyclin B verwendet, um eine gereinigte Ziel Protein in Vitrophosphorylieren. Phosphorylierung eines Substrates erhöht seine Masse und fügt zwei negative Ladungen; daher bestätigt erfolgreiche Phosphorylierung eine aufwärts Verschiebung der Protein Gel Band auf SDS-PAGE. Cdk1-spezifische Phosphorylierung Websites werden anschließend durch Massenspektrometrie Analyse des Proteins in Vitro phosphoryliert identifiziert. Experimentelles Design unterstützen, überprüfen wir auch Berechnungswerkzeuge und Referenzen für die Vorhersage von Cdk1-spezifische Phosphorylierung Sites die Proteinsequenz. Darüber hinaus erläutern wir im folgenden Reinigung-Protokolle, die sehr reine und homogene Protein Vorbereitungen für die Kinase Assay ergeben. Schließlich identifizierte Phosphorylierung Websites durch funktionelle Studien überprüft werden müssen, und eine einfache Bindung-Assay wird hier beschrieben, für diesen Zweck. Kombiniert, ist dies ein sehr leistungsfähiges Ansatz, der Renditen Cdk1-spezifische Phosphorylierung Sites und mechanistische Studien in wie Cdk1 den Zellzyklus7,10,11 steuert. Da diese Methode auf gereinigten Proteinen angewiesen ist, kann es zu jedem Modell Organismus und Erträge zuverlässige Ergebnisse angewendet werden. Funktionale Überprüfung des erhaltenen Phosphorylierung Websites in Vitro wird jedoch empfohlen, wie Zellen zusätzliche regulatorische Mechanismen, z. B. posttranslationale Modifikationen, Interaktionspartner oder zellulären Lokalisation verfügen, kann Phosphorylierung Websites oder unzugänglich für die Anerkennung von Cdk1 rendern.
Cdk1 erkennt eine Konsens-Phosphorylierung-Website, die besteht aus (Ser/Thr-Pro-X-Lys/Arg), wo X ist keine Rückstände und ein Serin oder Threonin der Ort der Phosphorylierung ist. Besonders wichtig für die Anerkennung ist das Vorhandensein von Prolin in der + 1-Position. Darüber hinaus werden grundlegende Rückstände in den + 2 oder + 3 Positionen bevorzugt, mit die meisten Cdk1-spezifische Phosphorylierung Websites mit einer Lys oder Arg auf die + 3 position6,12.
Aktivierung des Cdk1 ist streng geregelt und führt zu Beginn der Mitose1,2,3,4,5. Im Allgemeinen hängt die Aktivität von cyclin-abhängigen Kinasen ihre Assoziation mit deutlichen Cyclin (cyclin A, B, C, D und E in den Menschen), die bei oszillierenden Ebenen in der gesamten Zellzyklus13ausgedrückt werden. Cdk1 Ausdruck ist konstant über den Zellzyklus und die Regulierung ihrer Tätigkeit stützt sich auf seine Assoziation mit den regulatorischen Untereinheiten cyclin A und cyclin B5,13,14,15, als gut als Post-translationalen Modifikationen. Bildung des Cdk1/cyclin B-Komplex ist erforderlich für die Kinase Aktivierung5,14,15,16,17,18. In der G2-Phase cyclin B übersetzt in das Zytosol und importiert in den Zellkern, wo es an Cdk15,14,15,16,17,18 bindet; jedoch wird Cdk1/cyclin B inaktiviert durch Phosphorylierung an Thr14 und Tyr15 Rückstände von der menschlichen Cdk1-hemmenden Kinasen Myt1 (cdc2-hemmenden membranassoziierten Tyrosin und Threonin-spezifische Kinase) und Wee1, bzw.19gehalten, 20,21. In der Spätphase der G2 Dephosphorylation Thr14 und Tyr15 durch Zellteilung Zyklus 25 Phosphatase (cdc25) aktiviert die Kinase-Aktivität des Cdk1/cyclin B-Komplex und löst die Einleitung der Mitose12,14, 18 , 20 , 22 , 23. Phosphorylierung des Thr161 auch für Cdk1/cyclin B Aktivierung ist erforderlich und wird durch Cdk7, die Aktivierung von Cdk-Kinase (CAK)18vermittelt. Abbau von cyclin B in Anaphase inaktiviert Cdk1, so dass Ausstieg aus Mitose24,25. Aktivierung des Cdk1/cyclin B wird daher ein komplizierter Prozess. Die hier vorgestellten Protokoll erfolgt mit handelsüblichen Cdk1/cyclin B. Während rekombinante Expression dieses Komplexes in Insektenzellen es aktiviert in Vivo durch endogene Kinasen14,20 ist und bleibt aktiv im gereinigten Zustand. Die daraus resultierende aktive, rekombinante menschliche Cdk1/cyclin B eignet sich für in-vitro- Kinase-Assays.
Hier beschreiben wir ein Protokoll für die Identifizierung von Cdk1-spezifische Phosphorylierung Websites in den menschlichen Zentromer Protein F (CENP-F)10. CENP-F ist ein Kinetochor-Protein, das befindet sich im Zellkern während der meisten Zeit der Interphase (G1 und S-Phase) und der Ausfuhr nach dem Zytosol in der G2 Phase26,27,28 in einer Cdk1-abhängigen Weise10, 11. nuklearen Lokalisierung durch einen zweiseitigen cNLS26gewährt wird. cNLSs werden von den Nukleartransporten Faktor Karyopherin α, erkannt, die zusammen mit Karyopherin β und RanGDP, die Einfuhr von cNLS-Cargo in den Zellkern29erleichtert. Nuclear Export in der G2-Phase wird über eine unbekannte Export Weg10erleichtert. Sobald CENP-F in die Zellflüssigkeit befindet, es wird eingestellt, um die Kernhülle und wiederum stellt den motor Protein Komplex dynien30,31. Dieser Weg ist wichtig, den Kern der Centrosome entsprechend während der Anfangsphase der mitotischen Spindel Baugruppe in ein dynien-abhängigen Weise positionieren ist wichtig für das richtige Timing der mitotischen Eintrag und ein grundlegender Vorgang im Gehirn Entwicklung30,31,32. Beginnend in der G2-Phase, wird CENP-F auch in das Kinetochor eingebaut wo hat wichtige Aufgaben für Treue Chromosom Abtrennung27,28,33,34,35 . Ein wichtigster regulatorische Schritt dieser Wege ist der nuklearen Export von CENP-F in der G2-Phase, die Cdk110,11abhängig ist. Wir beschreiben hier ein Protokoll für die Identifizierung von Cdk1-spezifische Phosphorylierung Websites in die cNLS CENP-f Phosphomimetic Mutationen dieser Seiten verlangsamen nuclear Import von CENP-F, was darauf hindeutet, dass Cdk1/cyclin B direkt zellulären Lokalisation der CENP-F durch Phosphorylierung von seinem cNLS10regelt.
Insgesamt, dieser in-vitro- Kinase Assay ermöglicht die Identifikation von spezifischen Substraten für die Kinase Cdk1. Purified Zielproteine phosphorylierten in Vitro durch die im Handel erhältlichen Cdk1/cyclin B-Komplex und die Phosphorylierung Websites sind werden anschließend durch Massenspektrometrie identifiziert. Die Identifikation des Cdk1-spezifische Phosphorylierung Sites unterstützt mechanistische Studien, die zeigen, wie Cdk1 Zellzyklus steuert.

<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-abhängige Kinase 1 (Cdk1) ist ein master-Controller für den Zellzyklus in allen Eukaryoten und schätzungsweise 8-13 % des Proteoms phosphorylates; die Anzahl der identifizierten Ziele für Cdk1, besonders in den menschlichen Zellen ist jedoch nach wie vor niedrig. Die Identifizierung von Cdk1-spezifische Phosphorylierung Websites ist wichtig, da sie mechanistische Einblicke in wie Cdk1 Zellzyklus steuert. Regulierung des Zellzyklus ist entscheidend für die treuen Chromosom Abtrennung und Mängel in diesen komplizierten Prozess zu Chromosomenaberrationen und Krebs führen.
Hier beschreiben wir einen in-vitro- Kinase Assay, der verwendet wird, um Cdk1-spezifische Phosphorylierung Sites zu identifizieren. In diesem Test ist ein gereinigtes Protein phosphorylierten in Vitro kommerziell verfügbare menschliche Cdk1/cyclin B. erfolgreich Phosphorylierung wird bestätigt durch SDS-PAGE und Phosphorylierung Websites werden anschließend durch Massenspektrometrie identifiziert. Wir beschreiben auch Reinigung-Protokolle, die Ausbeute sehr reine und homogene Protein Vorbereitungen für die Kinase Assay und eine verbindliche Assay für die funktionale Verifikation der identifizierten Phosphorylierung-Standorte, die die Interaktion zwischen den Sonden ein klassischer nuklearen Lokalisierung-Signal (cNLS) und seine Nukleartransporten Rezeptor Karyopherin α. Experimentelles Design unterstützen, überprüfen wir Ansätze für die Vorhersage von Cdk1-spezifische Phosphorylierung Sites von Proteinsequenzen. Zusammen stellen diese Protokolle einen sehr leistungsfähigen Ansatz, der Renditen Cdk1-spezifische Phosphorylierung Sites und mechanistische Studien in wie Cdk1 Zellzyklus steuert. Da diese Methode stützt sich auf gereinigten Proteinen, kann es zu jedem Modell Organismus und Erträge zuverlässige Ergebnisse, vor allem in Kombination mit Zelle funktionelle Studien angewendet werden.

Wir haben vor kurzem einen in-vitro- Kinase Assay (Abbildung 1) verwendet, um Cdk1-spezifische Phosphorylierung Seiten in einem Fragment CENP-F zu ermitteln, die ein cNLS10enthalten. Dieses Signal verleiht nuklearen Lokalisierung von CENP-F während des größten Teils der Interphase. In der G2-Phase ist CENP-F aus dem Zellkern in das Zytosol in gewissem Sinne Cdk1-abhängige exportiert. Mechanistische Einblicke, wie Cdk1 rege...

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-abhängige Kinase 1 (Cdk1) wird in der G2-Phase des Zellzyklus aktiviert und reguliert viele zelluläre Signalwege. Hier präsentieren wir ein Protokoll für eine in-vitro- Kinase Assay mit Cdk1, wodurch die Identifizierung von Cdk1-spezifische Phosphorylierung Websites für zellulare Ziele von diesem wichtigen Kinase.

Identifizierung von Cyclin-abhängige Kinase 1 spezifische Phosphorylierung Websites durch eine 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

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

18.5K Aufrufe.  State University of New York at Binghamton. Cyclin-abhängige Kinase 1 (Cdk1) wird in der G2-Phase des Zellzyklus aktiviert und reguliert viele zelluläre Signalwege. Hier präsentieren wir ein Protokoll für eine in-vitro- Kinase Assay mit Cdk1, wodurch die Identifizierung von Cdk1-spezifische Phosphorylierung Websites für zellulare Ziele von diesem wichtigen Kinase.

Serie: 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.

Test der Proteinkinase-Aktivität mit radioaktiv markiertem 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 ...

Forschung

Biochemie

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.

Oligopeptid-Konkurrenz-Assay zur Phosphorylierungsbestimmung

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.

Identifizierung von neuartigen CK2 Kinase Substrate mit einem vielseitigen biochemischen Ansatz

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.

Charakterisierung auf molekularer Ebene mit robusten biochemischen Ansätzen eines neuen Kinase-Proteins

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.

Überwachung Kinase und Phosphatase-Aktivitäten durch den Zellzyklus durch Ratiometric FRET

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

Biologie

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.

Untersuchen der Kinase-Aktivität von LRRK2 In-vitro-</em

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.

Bezeichnung des Post-translationale Modifikationen von Pflanzenproteinkomplexe

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.

Identifikation von Kinase-Substrat-Pairs Mit 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 ...

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.

Identifizierung von Cyclin-abhängige Kinase 1 spezifische Phosphorylierung Websites durch eine In-vitro- Kinase Assay

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