Determination of substrate motifs for human Chk1 and hCds1/Chk2 by the oriented peptide library approach.
Mammalian Chk1 and Chk2 are two Ser/Thr effector kinases that play critical roles in DNA damage-activated cell cycle checkpoint signaling pathways downstream of ataxia telangiectasia-mutated and ataxia telangiectasia-related. Endogenous substrates have been identified for human hCds1/Chk2 and Chk1; however, the sequences surrounding the substrate residues appear unrelated, and consensus substrate motifs for the two Ser/Thr kinases remain unknown. We have utilized peptide library analyses to develop specific, highly preferred substrate motifs for hCds1/Chk2 and Chk1. The optimal motifs are similar for both kinases and most closely resemble the previously identified Chk1 and hCds1/Chk2 substrate target sequences in Cdc25C and Cdc25A, the regulation of which plays an important role in S and G(2)M arrest. Essential residues required for the definition of the optimal motifs were also identified. Utilization of the peptides to assay the substrate specificities and catalytic activities of Chk1 and hCds1/Chk2 revealed substantial differences between the two Ser/Thr kinases. Structural modeling analyses of the peptides into the Chk1 catalytic cleft were consistent with Chk1 kinase assays defining substrate suitability. The library-derived substrate preferences were applied in a genome-wide search program, revealing novel targets that might serve as substrates for hCds1/Chk2 or Chk1 kinase activity.
O'Neill
Ted
T
Center for Blood Research, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Giarratani
Lauren
L
Chen
Ping
P
Iyer
Lakshmanan
L
Lee
Chang-Hun
CH
Bobiak
Matthew
M
Kanai
Fumihiko
F
Zhou
Bin-Bing
BB
Chung
Jay H
JH
Rathbun
Gary A
GA
D000595:Amino Acid Sequence; D000818:Animals; D000071877:Checkpoint Kinase 1; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
C109294:HDS1 protein, Helix lucorum; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D019151:Peptide Library; D011994:Recombinant Proteins; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse