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 optimal 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.


CONCLUSIONS
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.
Mah
Angie S
AS
Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA. mah@caltech.edu
Elia
Andrew E H
AE
Devgan
Geeta
G
Ptacek
Jason
J
Schutkowski
Mike
M
Snyder
Michael
M
Yaffe
Michael B
MB
Deshaies
Raymond J
RJ
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D019151:Peptide Library; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D020543:Proteome / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
D019151:Peptide Library; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases