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

博士デビッド王、ハワード ヒューズ医学研究所、カリフォルニア大学バークレーは、質量分析および有用なコメントを感謝いたします。Xuelian 朱先生、上海、研究所生物科学、中国科学院、フルレングス CENP F 構造を提供するため、中国上海に感謝いたします。最後に、博士スーザンの命とり、博士ブライアン キャラハン博士クリストフ Grewer アット ビンガムトン ユニバーシティは、機器へのアクセスを感謝いたします。この研究は、ニューヨーク州立大学、ニューヨーク州立大学ビンガムトン校化学科研究財団によって賄われていた。

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私たちの in vitroのキナーゼ試金は、細胞周期のマスター コント ローラーであり多くの重要な細胞プロセスを調整するキナーゼ Cdk1 のための分子ターゲットを識別するために非常に強力な方法です。浄化された蛋白質が Cdk1 の基板は、でき特定のリン酸化部位の同定であるかどうか、方法が決まります。Cdk1 による燐酸化によってこれを容易に解明の細胞プロセスの規則のため。
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キナーゼは、ATP からのリン酸基を基板に転送し、多くの細胞プロセスを調整する酵素です。このリン酸化は可逆的で、高速、2 つの負電荷を追加しますと自由エネルギーを格納、セルで使用される最も一般的な修飾の一つです。Cdk1 はまたとして知られている細胞分裂周期蛋白質 2 相同物 (cdc2) マスター コント ローラーではすべて真核生物1,2,3,4、5、細胞周期では、廃止、プロテオーム6、7の推定 8 ~ 13%。
一方、最近のプロテオミクス研究は、タンパク質においてはほとんどの場合、多くのリン酸化サイトを識別したキナーゼのこれらの変更のための責任は不明です。ヒトの細胞、特に、知られている Cdk1 ターゲット数は低7です。Cdk1 が細胞周期を制御する方法を確立する解明が可能、Cdk1 固有のリン酸化部位の同定が重要です。細胞周期制御は忠実な染色体分配と細胞分裂のために重要、無数の細胞プロセスのこの重要な生理機能をサポートするために行う必要があります。これは転写や細胞構造と核膜、染色体凝縮と紡錘アセンブリの分解など、組織における劇的な再編と同様に有糸分裂、発症する前に翻訳を停止含まれています。規制緩和とこれらのプロセスでのエラーは、がん、先天異常、または有糸分裂細胞死を引き起こします。RO 3306 など Cdk1 の特異的阻害剤が開発8、機能研究の強力なツールを提供してこれらの阻害物質は、現在がん治療の臨床試験 (レビューの9を参照してください)。
ここでは、Cdk1 固有のリン酸化部位の同定を可能にする生体外でキナーゼ分析について述べる。このアッセイ市販人間 Cdk1/サイクリン B を使用して体外に精製されたターゲットのタンパク質をリン酸化します。基質のリン酸化その質量を増加され、2 つの負電荷;したがって、成功したリン酸化を SDS-PAGE の蛋白質ゲルのバンドの上方シフトを確認しました。Cdk1 固有のリン酸化サイトその後試験管内でリン酸化蛋白質の質量分析によって識別されます。実験計画を支援するためには、また計算のツールおよび蛋白質シーケンスから Cdk1 固有のリン酸化部位の予測を参照を確認します。さらに、高純度、均一なタンパク質製剤キナーゼのアッセイに最適をもたらすの浄化のプロトコルについても述べる。最後に、特定のリン酸化のサイトの機能の研究によって検証が必要、単純な結合の試金はその目的のためここに記載されています。組み合わせることで、これは Cdk1 のリン酸化サイトを生成して Cdk1 が細胞周期7,10,11を制御する方法に解明をしたりできるように非常に強力なアプローチです。このメソッドは、浄化された蛋白質に依存しているので、モデル有機体および利回り信頼性の高い結果に適用できます。得られるリン酸化サイト、体外の機能検証を推奨するただし、細胞局在や相互作用パートナー翻訳後修飾など、場所に追加的な規制メカニズムを持っていることアクセス可能または Cdk1 による認識のリン酸化サイトをレンダリング可能性があります。
Cdk1 は成っている (Ser/スレオニン-プロ-X-Lys/アルゼンチン)、X は任意の残基、セリンまたはトレオニンのリン酸化のサイトは、コンセンサスのリン酸化部位を認識しています。+1 の位置にプロリンの存在は認識のために特に重要です。また、塩基性残基、+2 や +3 位置に適して、+3 で Lys または Arg を含むほとんどの Cdk1 のリン酸化サイトを6,12の位置します。
Cdk1 活性化が堅く調整され、有糸分裂1,2,3,4、5の発症に 。サイクリン依存性キナーゼの活動は一般に振動する細胞周期13全体レベルで個別のサイクリン (サイクリン A、B、C、D、および E の人間) との関連付けに依存します。Cdk1 式細胞周期で一定であるし、サイクリン A ととして B5,13,14,15をサイクリン調節サブユニットとの関連付けに依存してその活動の規制同様のポスト翻訳の修正。Cdk1/サイクリン B の複合体の形成は、キナーゼ活性化5,14,15,16,17,18に必要です。G2 段階でサイクリン B は細胞質で翻訳され Cdk15,14,15,16,17,18; に結合核にインポートただし、Cdk1/サイクリン B は Myt1 人間 Cdk1 抑制キナーゼによって残基 Thr14 と Tyr15 のリン酸化 (膜準チロシン、スレオニン-固有抑制 cdc2 キナーゼ) と Wee1、それぞれ19、によって不活化保持されます。 20,21。遅い G2 段階で細胞分裂サイクル 25 ホスファターゼ (cdc25) による Thr14 と Tyr15 の脱燐酸化が Cdk1/サイクリン B の複合体のキナーゼ活動をアクティブにし、有糸分裂12,14,の開始をトリガー18,20,22,23. Thr161 リン酸化 Cdk1/サイクリン B の活性化にも必要ですし、Cdk 活性化キナーゼ (CAK)18Cdk7 によって媒介されます。後期ではサイクリン B の分解は、有糸分裂24,25からの出口を許可する Cdk1 を不活性化します。Cdk1/サイクリン B の活性化は、複雑なプロセスである従って。ここで提示されたプロトコルが市販 Cdk1/サイクリン B で実行されます。昆虫細胞で、この複合体の組み換え式の中にそれは活性生体内で内因性キナーゼ14,20清められた状態でアクティブなままになります。結果、組換え人間 Cdk1/サイクリン B キナーゼ試金体外に適しています。
ここでは、ひと動原体タンパク質 F (CENP F)10の Cdk1 固有のリン酸化部位の同定のためのプロトコルについて述べる。CENP F (G1、S 期) 中間期のほとんどの間、核に存在すると Cdk1 依存方法10, G2 の相26,27,28に細胞質にエクスポートは、動原体タンパク質である11. 核局在化二部 cNLS26によって授与されます。cNLSs は、karyopherin β と RanGDP、cNLS 貨物の核29へのインポートを容易にする核輸送因子 karyopherin α によって認識されます。G2 段階で核の輸出は、不明な輸出経路10を介して促進されます。CENP F は、細胞質に存在する、それが核封筒に募集、順番モーター蛋白質ダイニンの複雑な30,31を募集します。この経路は、有糸分裂開始のタイミングが正しいと脳で根本的なプロセスのために重要であるダイニン依存方法で紡錘アセンブリの最初の段階の間に中心体にそれぞれの核を配置することが重要開発30,31,32。G2 段階で開始、CENP F も完成、動原体に忠実な染色体分離27,28,33,34,35 に重要な役割をしています。.これらの経路の重要な規制ステップは Cdk110,11に依存している G2 段階で CENP F の核輸出です。CENP F の cNLS で Cdk1 固有のリン酸化部位の同定のためのプロトコルをご紹介します。これらのサイトの Phosphomimetic 突然変異は Cdk1/サイクリン B は、直接、cNLS10のリン酸化による CENP F の局在を調節することを示唆している CENP-F の核輸入に遅くなります。
全体的にみて、キナーゼ Cdk1。 精製された標的タンパク質リン酸化は体外市販 Cdk1/サイクリン B 複合体とリン酸化のサイトでこの体外のキナーゼ試金の特定の基板の識別を可能します。その後、質量分析によって識別されます。Cdk1 固有のリン酸化部位の同定は、Cdk1 が細胞周期を制御する方法を明らかにする機構の研究をサポートしています。

<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

サイクリン依存性キナーゼ 1 (Cdk1) は、すべての真核生物の細胞周期のマスター コント ローラー; プロテオームの推定 8 ~ 13% を廃止ただし、ひと細胞で特に Cdk1 の特定のターゲットの数はまだ低いです。Cdk1 固有のリン酸化部位の同定は、機械論的洞察 Cdk1 が細胞周期を制御する方法を提供するように重要です。細胞周期制御は忠実な染色体分配に重要なこの複雑なプロセスの欠陥染色体異常やがんに 。
ここでは、Cdk1 のリン酸化サイトを識別するために使用される生体外でキナーゼ分析について述べる。このアッセイで浄化された蛋白質リン酸化培養では, SDS-PAGE による市販人間 Cdk1/サイクリン b. 成功によってリン酸化を確認し、質量分析法によるリン酸化のサイトの識別後。プローブ間の相互作用特定のリン酸化のサイトの機能の検証のキナーゼ試金および結合の試金の適切な高純度、均一なタンパク質製剤をもたらす浄化のプロトコルについても述べる古典的な核局在化信号 (cNLS) とその核輸送受容体 karyopherin α。実験計画を支援するためには、タンパク質から Cdk1 固有のリン酸化部位の予測のためのアプローチを確認します。一緒にこれらのプロトコルは Cdk1 のリン酸化サイトを生成でき、Cdk1 が細胞周期を制御する方法を解明する非常に強力なアプローチを提示します。このメソッドは浄化された蛋白質、細胞機能の研究と組み合わせる場合は特に任意モデル有機体そして利回り信頼性の高い結果に適用できます。

最近 cNLS10に含まれている CENP F フラグメントの Cdk1 のリン酸化サイトを識別するために、体外のキナーゼ試金 (図 1) を使いました。この信号は、中間期のほとんどの間に CENP F の核局在化を与えます。G2 段階で CENP F が Cdk1 依存的に細胞質、核からエクスポートされます。Cdk1 が CENP F の局在を制御する方法の力学的洞察?...

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

サイクリン依存性キナーゼ 1 (Cdk1) 細胞周期 G2 段階で活性化し、多くの細胞経路を調節します。この重要なキナーゼの細胞内標的を確立するため Cdk1 固有のリン酸化部位の同定を可能にする Cdk1 と体外キナーゼ試金のためのプロトコルを紹介します。

サイクリン依存性キナーゼ 1 の同定の in Vitroキナーゼによって特定のリン酸化のサイトの試金します。

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 San Sebastian

Research

JoVE Journal

Biochemistry

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

18.5K ビュー.  State University of New York at Binghamton. サイクリン依存性キナーゼ 1 (Cdk1) 細胞周期 G2 段階で活性化し、多くの細胞経路を調節します。この重要なキナーゼの細胞内標的を確立するため Cdk1 固有のリン酸化部位の同定を可能にする Cdk1 と体外キナーゼ試金のためのプロトコルを紹介します。

ビデオ: 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.

放射性標識ATPによるタンパク質キナーゼ活性のアッセイ

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 ...

生化学

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.

リン酸化部位決定のためのオリゴペプチド競合アッセイ

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.

多彩な生化学的アプローチを用いた新規 CK2 キナーゼ基板の識別

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.

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

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.

FRETはレシオメトリックで細胞周期のキナーゼおよびホスファターゼの活動を監視

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

生物学

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.

LRRK2のキナーゼ活性を測定する in vitroで</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.

植物タンパク質複合体の翻訳後修飾の同定

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.

ハイスループットスクリーニングを使用したキナーゼ基質ペアの同定


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.

サイクリン依存性キナーゼ 1 の同定の in Vitroキナーゼによって特定のリン酸化のサイトの試金します。

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