Book chapter
Identification of Novel Substrates of MAP Kinase Cascades Using Bioengineered Kinases that Uniquely Utilize Analogs of ATP to Phosphorylate Substrates
MAP Kinase Signaling Protocols, pp.167-183
Methods in Molecular Biology, Humana Press Inc
01/01/2010
DOI: 10.1007/978-1-60761-795-2_10
PMID: 20811983
Abstract
The Mitogen-Activated Protein Kinase (MAPK) family of signaling molecules regulates a number of cellular processes through the direct phosphorylation and regulation of a plethora of cellular proteins. Identifying the direct substrates of the MAPK pathway proteins is important for determining how the effects of MAPK activation have such profound effects on cell biology. In this chapter, we describe one method for specific labeling and identification of direct MAPK substrates. A single or double point mutation is generated within the ATP binding domain at a particular residue(s) termed the "gatekeeper" that comes into close contact with the N6 position of ATP. Most kinases contain an ammo acid larger than alanine at this position. Mutation of the residue(s) to glycine or alanine generates a "pocket" that allows the mutant kinase to bind and uniquely utilize an analog of ATP that contains a chemical substituent at the N6 position. When radiolabeled analog is added to the mutant kinase and a complex mixture of cellular proteins, the only proteins that become radiolabeled are direct substrates of the mutant kinase. To label biologically relevant substrates, we take advantage of the direct binding of MAPKs to their substrates. An epitope tagged mutant kinase is expressed in cells and immunoprecipitated with associated substrates, which are then radiolabeled in an in vitro kinase reaction using (gamma-P-32) ATP analog. Larger, unlabeled kinase reactions are run in parallel and used to identify the substrates by mass spectrometry.
Details
- Title: Subtitle
- Identification of Novel Substrates of MAP Kinase Cascades Using Bioengineered Kinases that Uniquely Utilize Analogs of ATP to Phosphorylate Substrates
- Creators
- Hui Zheng - Medical University of South CarolinaAdnan Al-Ayoubi - Med Univ S Carolina, Dept Cell & Mol Pharmacol & Expt Therapeut, Charleston, SC 29425 USAScott T. Eblen - Medical University of South Carolina
- Contributors
- R Seger (Editor)
- Resource Type
- Book chapter
- Publication Details
- MAP Kinase Signaling Protocols, pp.167-183
- Publisher
- Humana Press Inc; TOTOWA
- Series
- Methods in Molecular Biology
- DOI
- 10.1007/978-1-60761-795-2_10
- PMID
- 20811983
- eISSN
- 1940-6029
- ISSN
- 1064-3745
- Number of pages
- 17
- Language
- English
- Date published
- 01/01/2010
- Academic Unit
- Cardiothoracic Surgery
- Record Identifier
- 9984701725902771
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