The Wip1 Phosphatase PPM1D Dephosphorylates SQ/TQ Motifs in Checkpoint Substrates Phosphorylated by PI3K-like Kinases

The wild-type p53-induced phosphatase Wip1 (PP2Cδ or PPM1D) is a member of the protein phosphatase 2C (PP2C) family and controls cell cycle checkpoints in response to DNA damage. p38 MAPK and ATM were identified as physiological substrates of Wip1, and we previously reported a substrate motif that w...

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Bibliographic Details
Published in:Biochemistry (Easton) 2007-11, Vol.46 (44), p.12594-12603
Main Authors: Yamaguchi, Hiroshi, Durell, Stewart R, Chatterjee, Deb K, Anderson, Carl W, Appella, Ettore
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Sequence
Ataxia Telangiectasia Mutated Proteins
Catalytic Domain - physiology
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - metabolism
DNA-Binding Proteins - metabolism
Humans
Models, Molecular
p38 Mitogen-Activated Protein Kinases - metabolism
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Phosphatidylinositol 3-Kinases - chemistry
Phosphatidylinositol 3-Kinases - metabolism
Phosphoprotein Phosphatases - chemistry
Phosphoprotein Phosphatases - metabolism
Phosphoprotein Phosphatases - physiology
Phosphorylation
Protein Phosphatase 2C
Protein-Serine-Threonine Kinases - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Substrate Specificity
Tumor Suppressor Proteins - metabolism
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Summary:The wild-type p53-induced phosphatase Wip1 (PP2Cδ or PPM1D) is a member of the protein phosphatase 2C (PP2C) family and controls cell cycle checkpoints in response to DNA damage. p38 MAPK and ATM were identified as physiological substrates of Wip1, and we previously reported a substrate motif that was defined using variants of the p38(180pT 182pY) diphosphorylated peptide, TDDEMpTGpYVAT. However, the substrate recognition motifs for Wip1 have not been fully defined as the sequences surrounding the targeted residues in ATM and p38 MAPK appear to be unrelated. Using a recombinant human Wip1 catalytic domain (rWip1), in this study we measured the kinetic parameters for variants of the ATM(1981pS) phosphopeptide, AFEEGpSQSTTI. We found that rWip1 dephosphorylates phosphoserine and phosphothreonine in the p(S/T)Q motif, which is an essential requirement for substrate recognition. In addition, acidic, hydrophobic, or aromatic amino acids surrounding the p(S/T)Q sequence have a positive influence, while basic amino acids have a negative influence on substrate dephosphorylation. The kinetic constants allow discrimination between true substrates and nonsubstrates of Wip1, and we identified several new putative substrates that include HDM2, SMC1A, ATR, and Wip1 itself. A three-dimensional molecular model of Wip1 with a bound substrate peptide and site-directed mutagenesis analyses suggested that the important residues for ATM(1981pS) substrate recognition are similar but not identical to those for the p38(180pT 182pY) substrate. Results from this study should be useful for predicting new physiological substrates that may be regulated by Wip1 and for developing selective anticancer drugs.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi701096s