Chemical Genetic Analysis of Protein Kinase Function in Plants
Identification of protein kinase targets and specific inhibition of individual kinase isoforms on the protein level in planta are important techniques to elucidate signal transduction pathways. The use of ATP-binding pocket mutants, the so-called gatekeep
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. Introduction The attribution of individual protein kinases to specific signaling pathways and phosphorylation of substrates is often hampered by the sheer number of protein kinases and phosphorylation events present in the cell. Phosphorylation is probably the most universal and abundant posttranslational modification in proteins. In eukaryotic cells, about one third of all proteins are phosphorylated (1) with 99% of all phosphorylations appearing on serine and threonine residues (2). Current annotations of the Arabidopsis thaliana genome account for 1,003 proteins harboring a protein kinase domain (Pfam protein family database code: PF00069), representing about 4% of all Arabidopsis proteins. In vitro protein substrate labeling using radioactive [g-32P]-ATP therefore produces a high background of phosphorylation by other kinases, making it difficult N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_15, © Springer Science+Business Media, LLC 2011
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to detect specific changes in phosphorylation patterns. Pharmacological inhibition of kinases is also hindered by low specificity of kinase inhibitors, preventing analysis of individual kinase isoforms. A major breakthrough in the development of specific kinase inhibitors was the introduction of the “chemical genetics” approach, which was developed and refined by Kevan Shokat and co-workers (3–9). It is based on the genetic engineering of protein kinases to introduce specific amino acid substitutions into the conserved ATP-binding pocket to accommodate modified nucleotides and kinase inhibitors that carry bulky side chains. Since these modified substrates and inhibitors are not recognized by wild-type kinases this approach allows specific inhibition of individual kinases and even of specific isoforms in vivo without interfering with all other signaling kinases. In the original study, two residues of the ATPbinding pocket of the viral kinase v-Src (Val323 and Ile338) are identified that lie in close vicinity (5 Å) to the N6-amine of ATP (Table 1). It could be shown that a variant of this kinase, in which both of these residues are substituted by alanine, accepts the ATP analogue N6-cyclohexyl-ATP as phosphate donor (3). Further improvement of the method has shown that only the Ile338 residue controls binding of N6-modified nucleotides (10). Therefore, this residue has been termed “gatekeeper” residue.
Table 1 Structure-based sequence alignment of various kinases that have been modified for the chemical-genetic approach b-sheets Kinase
b2
b3
b4
References
v-Src
E V W
V A I K T
L
V323 Q
Y
I
V
I338
(3)
CDK2 (H.s.)
V V Y
V A L K K
I
V
K
Y
L V
F80
(8)
CamKIIa (H.s.)
V V R
Y
A A K I
I
V
R
Y
L I
F89
(6)
Fus3 (S.c.)
V V C
V A I K K
I
I
T
Y
I
I
Q93
(6)
Don3 (U.m.)
T V Y
V A I K Q
V T
R
W I
I
M157 (11)
MPK4 (A.t.)
I
V C
V A I K K
V I
A
Y
I
V
Y124 (12)
Pto (S.l.)
K V Y
V A L K R
L
V
S
V
L
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