Abstract
Engineered kinases and thiophosphate enrichment were used to identify many candidate CDK2 substrates in human cell lysates.
Highlights
Protein phosphorylation regulates a multitude of biological processes
We selected several candidate substrates and confirmed that they were phosphorylated by cyclin A-CDK2 in vitro on the same sites that we identified in the screen
Because the enzyme preparation we used in these studies contained an excess of free CDK2 (F80A), we considered the possibility that some substrate phosphorylations might result from the association of endogenous cyclins with CDK2 (F80A) that was either monomeric, or that may have dissociated from cyclin A during the assay conditions
Summary
Protein phosphorylation regulates a multitude of biological processes. the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. Genome Biology 2008, Volume 9, Issue 10, Article R149 Chi et al R149.2 in vivo, but this information is critical to understanding kinase functions and the control of biological processes in general. It is difficult to apply these approaches to kinases that phosphorylate broader substrate motifs, since there is less epitope conservation among substrates. Another recent method combined quantitative phosphoproteomics with kinase knock-outs and cellular perturbations to identify kinase targets in yeast [5]. With these cell-based approaches, it is often difficult to determine if the putative substrates are direct kinase targets. An in vitro approach employing arrays of proteins phosphorylated by isolated recombinant kinases has been successfully used in a global analysis of yeast kinase substrates, but this strategy may be difficult to apply to organisms with larger proteomes [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
