Substrate specificity of the human protein phosphatase 2C delta 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 p38 alpha and in a new substrate, UNG2. rWip1 showed properties that were comparable to those of PP2C alpha or full-length Wip1 in terms of affinity for Mg2+, 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 PP2C alpha 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

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