Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates
Article first published online: 26 JAN 2006
The Plant Journal
Volume 45, Issue 4, pages 512–522, February 2006
How to Cite
Peck, S. C. (2006), Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates. The Plant Journal, 45: 512–522. doi: 10.1111/j.1365-313X.2005.02613.x
- Issue published online: 26 JAN 2006
- Article first published online: 26 JAN 2006
- Received 7 June 2005; revised 12 August 2005; accepted 31 August 2005.
- signal transduction;
- in vitro kinase assay;
- phosphorylation site analysis;
- phosphatase assay;
- mitogen-activated protein kinase
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.