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.
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA.
Janine
J
Mok
Philip M
PM
Kim
Hugo Y K
HY
Lam
Stacy
S
Piccirillo
Xiuqiong
X
Zhou
Grace R
GR
Jeschke
Douglas L
DL
Sheridan
Sirlester A
SA
Parker
Ved
V
Desai
Miri
M
Jwa
Elisabetta
E
Cameroni
Hengyao
H
Niu
Matthew
M
Good
Attila
A
Remenyi
Jia-Lin Nianhan
JL
Ma
Yi-Jun
YJ
Sheu
Holly E
HE
Sassi
Richelle
R
Sopko
Clarence S M
CS
Chan
Claudio
C
De Virgilio
Nancy M
NM
Hollingsworth
Wendell A
WA
Lim
David F
DF
Stern
Bruce
B
Stillman
Brenda J
BJ
Andrews
Mark B
MB
Gerstein
Michael
M
Snyder
Benjamin E
BE
Turk
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