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Open Access Highly Accessed Research article

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

Angie S Mah1, Andrew EH Elia34, Geeta Devgan6, Jason Ptacek7, Mike Schutkowski8, Michael Snyder67, Michael B Yaffe345 and Raymond J Deshaies12*

Author Affiliations

1 Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA

2 Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA

3 Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

4 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

5 Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

6 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA

7 Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520, USA

8 JPT Peptide Technologies GmbH, Invalidenstrasse 130, 10115 Berlin, Germany, USA

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BMC Biochemistry 2005, 6:22  doi:10.1186/1471-2091-6-22

Published: 21 October 2005

Abstract

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