The Wip1 phosphatase PPM1D dephosphorylates SQ/TQ motifs in checkpoint substrates phosphorylated by PI3K-like kinases.
The wild-type p53-induced phosphatase Wip1 (PP2Cdelta 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.
Yamaguchi
Hiroshi
H
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Durell
Stewart R
SR
Chatterjee
Deb K
DK
Anderson
Carl W
CW
Appella
Ettore
E
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D020134:Catalytic Domain / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D010766:Phosphorylation; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D048051:p38 Mitogen-Activated Protein Kinases; C576324:ATM protein, human; C000607456:PPM1A protein, human; C000607457:PPM1B protein, human; C000607458:PPM1D protein, human; C000607460:PPM1G protein, human