Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates.
Protein phosphorylation is a highly conserved mechanism for regulating protein function, being found in all prokaryotes and eukaryotes examined. Phosphorylation can alter protein activity or subcellular localization, target proteins for degradation and effect dynamic changes in protein complexes. In many cases, different kinases may be involved in each of these processes for a single protein, allowing a large degree of combinatorial regulation at the post-translational level. Therefore, knowing which kinases are activated during a response and which proteins are substrates is integral to understanding the mechanistic regulation of a wide range of biological processes. In this paper, I will describe methods for monitoring kinase activity, investigating kinase-substrate specificity, examining phosphorylation in planta and the determination of phosphorylation sites in a protein. In addition, strategic considerations for experimental design and variables will be discussed.
Peck
Scott C
SC
University of Missouri-Columbia, 271H Life Sciences Center, Columbia, MO 65211, USA. pecks@missouri.edu
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010944:Plants / Q000201:enzymology / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases