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Published October 21, 2005 | Published
Journal Article Open

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening


Background: The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified. Results: Using a combination of peptide library selection, phosphorylation of opitmal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis. Conclusion: We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site.

Additional Information

© 2005 Mah et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 11 June 2005; Accepted: 21 October 2005; Published: 21 October 2005. Authors' contributions: ASM performed the peptide kinase assays and analysis, the immunoprecipitations and kinase assays of the TAP-tagged strains, preparation of recombinant Dbf2-Mob1 complexes used throughout the work, and preparation of the manuscript. AEHE carried out the peptide library screening and analysis. GD and JP carried out the proteome chip studies. MS performed the phosphosite array screening. MS, MBY, and RJD contributed to the experimental design, analysis, and interpretation. Acknowledgements: We thank Ramzi Azzam for his invaluable insight and enthusiasm for initiating this project. We also thank Dane Mohl and William Ja for their thoughts and comments on this work. We are also grateful to Heng Zhu for performing initial proteome chip experiments and Gary Kleiger for providing bioinformatics expertise. This research was supported by an NIH grant to RJD (GM059940). http://www.biomedcentral.com/1471-2091/6/22

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