Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs.
Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P-3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.
Mok
Janine
J
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA.
Kim
Philip M
PM
Lam
Hugo Y K
HY
Piccirillo
Stacy
S
Zhou
Xiuqiong
X
Jeschke
Grace R
GR
Sheridan
Douglas L
DL
Parker
Sirlester A
SA
Desai
Ved
V
Jwa
Miri
M
Cameroni
Elisabetta
E
Niu
Hengyao
H
Good
Matthew
M
Remenyi
Attila
A
Ma
Jia-Lin Nianhan
JL
Sheu
Yi-Jun
YJ
Sassi
Holly E
HE
Sopko
Richelle
R
Chan
Clarence S M
CS
De Virgilio
Claudio
C
Hollingsworth
Nancy M
NM
Lim
Wendell A
WA
Stern
David F
DF
Stillman
Bruce
B
Andrews
Brenda J
BJ
Gerstein
Mark B
MB
Snyder
Michael
M
Turk
Benjamin E
BE
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity
D011494:Protein Kinases