Substrate specificity of the human protein phosphatase 2Cdelta, Wip1.
Wip1, the wild-type p53-induced phosphatase, selectively dephosphorylates a threonine residue on p38 MAPK and mediates a negative feedback loop of the p38 MAPK-p53 signaling pathway. To identify the substrate specificity of Wip1, we prepared a recombinant human Wip1 catalytic domain (rWip1) and measured kinetic parameters for phosphopeptides containing the dephosphorylation sites in p38alpha and in a new substrate, UNG2. rWip1 showed properties that were comparable to those of PP2Calpha or full-length Wip1 in terms of affinity for Mg(2+), insensitivity to okadaic acid, and threonine dephosphorylation. The substrate specificity constant k(cat)/K(m) for a diphosphorylated peptide with a pTXpY sequence was 6-8-fold higher than that of a monophosphorylated peptide with a pTXY sequence, while PP2Calpha showed a preference for monophosphorylated peptides. Although individual side chains before and after the pTXpY sequence of the substrate did not have a significant effect on rWip1 activity, a chain length of at least five residues, including the pTXpY sequence, was important for substrate recognition by rWip1. Moreover, the X residue in the pTXpY sequence affected affinity for rWip1 and correlated with selectivity for MAPKs. These findings suggest that substrate recognition by Wip1 is centered toward a very narrow region around the pTXpY sequence. Three-dimension homology models of Wip1 with bound substrate peptides were constructed, and site-directed mutagenesis was performed to confirm the importance of specific residues for substrate recognition. The results of our study should be useful for predicting new physiological substrates and for designing specific Wip1 inhibitors.
Yamaguchi
Hiroshi
H
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Minopoli
Giuseppina
G
Demidov
Oleg N
ON
Chatterjee
Deb K
DK
Anderson
Carl W
CW
Durell
Stewart R
SR
Appella
Ettore
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D000071636:Protein Phosphatase 2C; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D017931:DNA Primers; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D019319:Okadaic Acid; D013912:Threonine; C000607458:PPM1D protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C