עבודה זו נתמכה על ידי הקרן הלאומית למחקר של קוריאה מענק ממומן על ידי ממשלת קוריאה (MSIP) (מס &#39;2015R1A2A1A10052663 ו No. 2014M1A3A3A02034698).

<ol><li>Hardie, D. G., Schaffer, B. E., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPK%3A+An+energy-sensing+pathway+with+multiple+inputs+and+outputs%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">AMPK: An energy-sensing pathway with multiple inputs and outputs.</a> Trends Cell Biol. 26, 190-201 (2016).</li>
<li>Gwinn, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPK+phosphorylation+of+raptor+mediates+a+metabolic+checkpoint%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">AMPK phosphorylation of raptor mediates a metabolic checkpoint.</a> Mol Cell. 30, 214-226 (2008).</li>
<li>Huang, H. C., Nguyen, T., Pickett, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorylation+of+Nrf2+at+Ser-40+by+protein+kinase+C+regulates+antioxidant+response+element-mediated+transcription%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.</a> J Biol Chem. 277, 42769-42774 (2002).</li>
<li>Niture, S. K., Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antioxidant-induced+modification+of+INrf2+cysteine+151+and+PKC-delta-mediated+phosphorylation+of+Nrf2+serine+40+are+both+required+for+stabilization+and+nuclear+translocation+of+Nrf2+and+increased+drug+resistance%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Antioxidant-induced modification of INrf2 cysteine 151 and PKC-delta-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance.</a> J Cell Sci. 122, 4452-4464 (2009).</li>
<li>Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GSK-3beta+acts+upstream+of+Fyn+kinase+in+regulation+of+nuclear+export+and+degradation+of+NF-E2+related+factor+2%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2.</a> J Biol Chem. 282, 16502-16510 (2007).</li>
<li>Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorylation+analysis+by+mass+spectrometry%3A+myths%2C+facts%2C+and+the+consequences+for+qualitative+and+quantitative+measurements%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.</a> Mol Cell Proteomics. 5, 172-181 (2006).</li>
<li>Dephoure, N., Gould, K. L., Gygi, S. P., Kellogg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+and+analysis+of+phosphorylation+sites%3A+a+quick+guide+for+cell+biologists%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.</a> Mol Biol Cell. 24, 535-542 (2013).</li>
<li>Joo, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPK+facilitates+nuclear+accumulation+of+Nrf2+by+phosphorylating+at+Serine+550%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at Serine 550.</a> Mol Cell Biol. 36, 1931-1942 (2016).</li>
<li>Sinclair, W. K., Holloway, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Half-lives+of+some+radioactive+isotopes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Half-lives of some radioactive isotopes.</a> Nature. 167 (4244), 365(1951).</li>
<li>Chang, B. Y., Chiang, M., Cartwright, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interaction+of+Src+and+RACK1+is+enhanced+by+activation+of+protein+kinase+C+and+tyrosine+phosphorylation+of+RACK1%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1.</a> J Biol Chem. 276, 20346-20356 (2001).</li>
<li>Smith, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RACK1+interacts+with+filamin-A+to+regulate+plasma+membrane+levels+of+the+cystic+fibrosis+transmembrane+conductance+regulator%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.</a> Am J Physiol Cell Physiol. 305 (1), C111-C120 (2013).</li>
</ol>

למחברים אין מה לחשוף.

כדרך פשוטה ונוחה להעריך את האותנטיות של אתרי זרחון חזוי בתיווך AMPK, כאן אנו מתארים assay קינאז במבחנה שניתן להשתמש בהם כדי לגלות אתר זרחן ספציפי באמצעות פפטידים תחרותיים כדי לאמת אותו באמצעות מוטציה אתר ספציפי . הנתונים המייצגים המתקבלים מתוך מבחנה פעילות מ?...

חלבון זרחן ב שאריות מסוים ממלא תפקיד משמעותי במגוון רחב של תהליכים תאיים. לפיכך, הבנה של רשתות איתות דורשת זיהוי של אתרי זרחן ספציפיים. בנוסף, האתר זרחן קובע את ההשפעה על תפקוד החלבון, כי תחומים בודדים בתוך חלבון יש מבנים שונים פונקציות. פעילות הגורם הגרעיני erythroid 2 הקשורים גורם 2 (Nrf2), גורם נוגד חמצון מפתח, הוא מוסדר דו כיווני באמצעות זרחון באתרים שונים. המחקרים שלנו התמקדו קינאזות כי לזרז את זרחון של Nrf2. תגובת הלחץ של Nrf2 נגד אתגר חמצוני מתרחשת במהירות, בעיקר באמצעות זרחון ב serine 40 ו מתווכת על ידי חלבון קינאז C (PKC) -δ, אשר מפעיל Nrf2 3 , 4 . לעומת זאת, Fyn מזרז את זרחון מעכב של Nrf2 ב tyrosine 5עבור פיקוח הדוק על הפעילות. השיטה הנפוצה ביותר המשמשת לגלות אתרי זרחון הוא כרומטוגרפיה נוזלית / ספקטרומטריית מסה (LC / MS) ניתוח. מהיר וזמן רגיש זרחון באתר מיפוי נתונים ניתן להפיק בדרך זו; עם זאת, יש כמה מגבלות טכניות, לעתים קרובות יצירת אותות מזויפים שלילי. כיסוי רצף גרוע לעתים קרובות מתרחשת ניתוח LC / MS. כדי לזהות אתרי זרחון, מידע על כיסוי מקסימלי של חומצות אמינו של חלבון נדרש 6 . פרוטאוליזה של חלבון של עניין עם מספר פרוטאזות במהלך הצעד העיכול עשוי להיות לעזור בשיפור רצף הכיסוי. מכשול נוסף לזיהוי שאריות זרחון הוא אובדן של חומצה זרחתית כי הוא נצפה לעתים קרובות עבור serine ו threonine-phosphideslated פפטידים 6 , 7 . זרחן פוספט moieties הם לעתים קרובותשוחרר מ phosphopeptides במהלך תהליך הפיצול 7 . האפשרות השנייה בעת חיפוש אתרי זרחון היא באמצעות שיטת microarray פפטיד. ניתן לבדוק את אתרי היעד kinase באמצעות שבב microarray המכיל שברי פפטיד נגזר חלבון של עניין. עם זאת, בשל הדרישה הציוד לייצור וזיהוי שבב microarray, שיטת microarray פפטיד נחשב זמן רב ויקר. כדי להתגבר על אתגרים אלה, במבחנה oligopeptide- מתחרה assay קינאז יכול לשמש קינאז חלבון עם מוטיבים הכרה ידוע. אם מוטיב ההכרה של קונצנזוס של קינאז הוא הקימה, אתרי זרחון putative של המצע המועמד ניתן לחזות, ואת האותנטיות של האתרים ניתן לאמת. השיטה המשכנעת ביותר עבור הליך זה היא להראות את הביטול של זרחון בחלבון מוטנטי שבו תחזיתD שאריות מוחלפת בחומצת אמינו שאינה phosphorylatable ( כלומר, serine או threonine כדי alanine, tyrosine כדי פנילאלנין). עם זאת, ייצור ובידוד של חלבונים מוטנטים הוא זמן רב. כחלופה בשלב הראשוני של המחקר, assay פפטיד קינאז תחרותי הוא פשוט ונוח. כאן, אנו מתארים פרוטוקול עבור assay מבחנה במבחנה פפטיד ו עבור אימות של אתר זרחון.

<table><tbody><tr><td>HEPES</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>15630</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO</td><td>208337</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich, St. Louis, MO</td><td>E3889</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich, St. Louis, MO</td><td>D9779</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>G9422</td><td></td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO</td><td>450243</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Calbiochem, Nottingham, UK</td><td>539134</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A2383</td><td></td></tr><tr><td>AMP</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A1752</td><td></td></tr><tr><td>AMPK</td><td>Upstate Biotechnology, Lake Placid, NY</td><td>14-840</td><td></td></tr><tr><td>Nrf2 (WT)</td><td>Abnova, Taipei City, Taiwan</td><td>H00004780-P01</td><td></td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]-ATP</td><td>PerkinElmer Life and Analytical Sciences, Waltham, MA</td><td>NEG502A</td><td></td></tr><tr><td>EZblue staining reagent</td><td>Sigma-Aldrich, St. Louis, MO</td><td>G1041</td><td></td></tr><tr><td>Pfu turbo DNA polymerase</td><td>Agilent Technologies, Santa Clara, CA</td><td>600250</td><td></td></tr><tr><td>dNTP mix</td><td>Agilent Technologies, Santa Clara, CA</td><td>200415-51</td><td>Avoid multiple thaw and freezing cycle</td></tr><tr><td>DpnI</td><td>New England Biolabs, Ipswich, MA</td><td>R0176S</td><td></td></tr><tr><td>LB broth</td><td>Duchefa Biochemie BV, Haarlem, Netherlands</td><td>L1704</td><td></td></tr><tr><td>Ampicillin</td><td>Affymetrix, Santa Clara, CA</td><td>11259</td><td></td></tr><tr><td>Agarose LE</td><td>iNtRON Biotechnology, Sungnam, South Korea</td><td>32034</td><td></td></tr><tr><td>HiYield Plus Gel/PCR DNA Mini Kit</td><td>Real Biotech Corporation, Taipei, Taiwan</td><td>QDF100</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>161-0400</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Bovogen Biologicals, Victoria, Australia</td><td>BSA100</td><td></td></tr><tr><td>Glutathione Sepharose 4B</td><td>GE Healthcare, Marlborough, MA</td><td>17-0756-01</td><td></td></tr><tr><td>Acetic Acid glacial</td><td>Duksan pure chemicals, Ansan, South Korea</td><td></td><td></td></tr><tr><td>Methyl alcohol&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;</td><td>Daejung Chemicals &amp;amp; Metals, Siheung, South Korea</td><td>5558-4410</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Typhoon FLA 7000</td><td>GE Healthcare, Marlborough, MA</td><td>28-9558-09</td><td></td></tr><tr><td>SDS-PAGE kit</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>1658001FC</td><td></td></tr><tr><td>Vacuum pump</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>165-178</td><td></td></tr><tr><td>Gel dryer</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>165-1746</td><td></td></tr><tr><td>Dancing shaker</td><td>FINEPCR, Seoul, Korea</td><td>CR300</td><td>The machine is needed for washing step</td></tr><tr><td>PCR machine</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>T100</td><td></td></tr><tr><td>Incubator/shakers</td><td>N-BIOTEK, GyeongGi-Do, Korea</td><td>NB-205L</td><td></td></tr><tr><td>Microcentrifuges</td><td>LABOGENE, Seoul, Korea</td><td>1730R</td><td></td></tr><tr><td>Chromatography columns</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>732-1010</td><td></td></tr></tbody></table>

oligopeptide competition assay for phosphorylation site determination

זרחון חלבון באתרים ספציפיים קובע את הקונפורמציה שלו ואת האינטראקציה עם מולקולות אחרות. לפיכך, זרחון חלבון משפיע על פונקציות ביולוגיות ומאפיינים של התא. כיום, השיטה הנפוצה ביותר לגילוי אתרי זירחון היא ניתוח כרומטוגרפי נוזלי / ספקטרומטריית מסה (LC / MS), שיטה מהירה ורגישה. עם זאת, זרחני פוספט זולה יחסית משוחררים לעתים קרובות phosphopeptides במהלך שלב פיצול, אשר לעתים קרובות מניב אותות שלילי שווא. במקרים כאלה, מסורתי assay קינאז במבחנה באמצעות מוטנטים מכוונים האתר יהיה מדויק יותר, אבל שיטה זו היא מייגעת זמן רב. לכן, שיטה חלופית באמצעות תחרות פפטיד עשוי להיות יתרון. מוטיב ההכרה הקונצנזוס של 5 &#39;אדנוזין monophosphate המופעל קינאז חלבון (AMPK) הוקם 1 ו תוקף באמצעות פוזיטיון סריקה פפטיד סריקה התחתAy. לפיכך, אתרי זרחון AMPK עבור מצע הרומן יכול להיות ניבא ואושר על ידי מבחני התחרות פפטיד. בדו&quot;ח זה, אנו מתארים את השלבים המפורטים ונהלים עבור במבחנה oligopeptide- מתחרה assay קינאז ידי המחשה AMPK בתיווך גורם גרעיני erythroid 2 הקשורים גורם 2 (Nrf2) זרחון. כדי לאמת את האתר זרחן, ביצענו רציף assay קינאז במבחנה באמצעות מוטציה באתר ספציפי. בסך הכל, assay התחרות פפטיד מספק שיטה למסך אתרי זרחן פוטנציאליים פוטנציאליים כדי לזהות אתרים לאימות על ידי מוטציות האתר זרחן.

איור 1 ואיור 2 מדגימים את התוצאות מניסויים חוזרים ונשנים בעיתון שדווח בעבר. שלושה oligopeptides 10-שאריות שונים מחקה את אתרי היעד AMPK putative (# 1, 148-157 המורכב SR153, # 2, 330-339 המורכב SR335, ו # 3, 553-562 המורכבת Ser558) היו מסונתז המשמשים מתחרים ...

Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

Oligopeptide Competition Assay for Phosphorylation Site Determination

מבחני תחרות פפטיד נמצאים בשימוש נרחב במגוון של ניסויים מולקולריים וחיסונית. מאמר זה מתאר שיטה מפורטת עבור המבחנה oligopeptide- מתחרה assay קינאז ואת הליכי אימות הקשורים, אשר עשוי להיות שימושי כדי למצוא אתרי זרחן ספציפיים.

Oligopeptide Assay תחרות עבור זרחון קביעת האתר

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

oligopeptide-competition-assay-for-phosphorylation-site-determination

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.4K Views.  Seoul National University.  מבחני תחרות פפטיד נמצאים בשימוש נרחב במגוון של ניסויים מולקולריים וחיסונית. מאמר זה מתאר שיטה מפורטת עבור המבחנה oligopeptide- מתחרה assay קינאז ואת הליכי אימות הקשורים, אשר עשוי להיות שימושי כדי למצוא אתרי זרחן ספציפיים. 

Video: Oligopeptide Competition Assay for Phosphorylation Site Determination

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.

Assaying חלבון פעילות עם Kinase Radiolabeled

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 ...

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.

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.

זיהוי של קינאז תלוי-Cyclin 1 אתרי זירחון ספציפיים על-ידי קינאז במבחנה 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 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.

זיהוי של סובסטרטים קינאז CK2 הרומן תוך שימוש בגישה הביוכימי רב-תכליתי

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.

אפיון ברמה המולקולרית באמצעות גישות ביוכימיות איתנות של חלבון קינאז חדש

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

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

תרגום לא רדיואקטיבי למדוד פוספורילציה פולינוקלאוטיד של מצעי נוקלאוטיד קטנים

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

בדיקה ממוטבת של משיכה במולקולה בודדת לכימות של זרחון חלבונים

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Co-immunoprecipitation Assay ללמוד אינטראקציות פונקציונלי בין קולטנים ואנזימים

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

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.

זיהוי לאחר translational השינויים של קומפלקסי חלבוני צמח

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

Biology

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.

זיהוי של זוגות קינאז-מצע שימוש הקרנת תפוקה גבוהה


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

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

השוואת הזיקה של חלבונים מחייב GTPase באמצעות מבחני תחרות

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...