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Figure 1 | Genome Biology

Figure 1

From: A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1

Figure 1

Producing a Chk1 kinase derivative able to use N6-benzyl(N6B)-ATP. (a) Amino acid alignment of ATP-binding pockets of human Chk1, Saccharomyces cerevisiae Cdc28, human Cdk2 and c-Abl, and viral v-Src. The identified gatekeeper amino acids are highlighted. (b) ATP-binding pocket of Chk1 (based on PDB entry 1IA8) showing the position of the gatekeeper residue Leu84 (i), the L84G mutation (ii), or the L84G mutated ATP-binding pocket accommodating N6B-adenosine (iii). Models were drawn by Chimera software [60]. (c) Chk1-L84G can use ATP analogues. In vitro kinase assay using wild type (WT) or gatekeeper mutant versions (L84A, L84G) of Chk1 in the presence of ATP or N6B-ATP. Active kinases phosphorylate Cdc25A on Ser123 as detected by phospho-specific antibody. Chk1 mobility shift due to autophosphorylation is indicated by arrows; 0.5 μg of each recombinant protein was used. (d) Recombinant WT Chk1 autophosphorylates on Ser345 and Ser317 as detected by phospho-specific antibodies; 1 μg of recombinant Chk1 was used.

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