LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1)

LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.     

Arsenic trioxide augments Chk2/p53-mediated apoptosis by inhibiting oncogenic Wip1 phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.     

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.     

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G₁ phase to G₂ and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G₁ cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.     

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.     

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.     

Development of a substrate-based cyclic phosphopeptide inhibitor of protein phosphatase 2Cdelta, Wip1

The wild-type p53-induced phosphatase, Wip1 (PP2Cdelta or PPM1D) is a member of the protein phosphatase 2C (PP2C) family and functions as a negative regulator of the p38 MAP kinase-p53 signaling pathway. PPM1D is amplified or Wip1 is overexpressed in several human cancers, and it acts as a weak oncogene. Although inhibition of Wip1 may have therapeutic value, no specific inhibitors are available. In this study, we designed phosphopeptide inhibitors for Wip1 on the basis of its optimal substrate sequence. We found that phosphoserine-containing diphosphorylated peptides with the sequence pSXpY inhibited Wip1 phosphatase activity, whereas phosphothreonine-containing peptides with the sequence pTXpY were physiological substrates. Moreover, the X residue in the pSXpY sequence modulated inhibitor activity, and beta-branched amino acid-substituted (Ile or Val) phosphopeptides showed high inhibitory potencies. A thioether cyclic phosphopeptide c(MpSIpYVA) had a K(i) <1.0 microM. Two serine/threonine phosphatases, PP2Calpha and PP2A, were not significantly inhibited by the cyclic phosphopeptide with a nonhydrolyzable phosphoserine mimetic. A homology model of Wip1 bound to a cyclic phosphopeptide and site-directed mutagenesis helped to identify residues important for Wip1 inhibitor selectivity among the PP2C family. These results provide the first proof of concept of a specific inhibitor of the catalytic site of Wip1 and should be useful for developing potential anti-cancer drugs.     

Intrinsic kinase activity and SQ/TQ domain of Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for regulation of Chk2 by Wip1

The anti-oncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell cycle checkpoint regulation. Recently, we have shown that Chk2 associates with the oncogenic Wip1 (PPM1D) phosphatase and that Wip1 acts as a negative regulator of Chk2 during DNA damage response by dephosphorylating phosphorylated Thr-68 in activated Chk2 (Fujimoto, H., Onishi, N., Kato, N., Takekawa, M., Xu, X. Z., Kosugi, A., Kondo, T., Imamura, M., Oishi, I., Yoda, A., and Minami, Y. (2006) Cell Death Differ. 13, 1170-1180). Here, we performed structure-function analyses of Chk2 and Wip1 by using a series of deletion or amino acid-substituted mutant proteins of Chk2 and Wip1. We show that nuclear localization of both Chk2 and Wip1 is required for their association in cultured cells and that the serine-glutamine (SQ)/threonine-glutamine (TQ) domain of Chk2, containing Thr-68, and the N-terminal domain of Wip1, comprising about 100 amino acids, are necessary and sufficient for the association of both molecules. However, it was found that an intrinsic kinase activity of Chk2, but not phosphatase activity of Wip1, is required for the association of fulllength Chk2 and Wip1. Interestingly, we also show that the mutant Wip1 proteins, bearing the N-terminal domain of Wip1 alone or lacking an intrinsic phosphatase activity, exhibit dominant negative effects on the functions of the wild-type Wip1, i.e. ectopic expression of either of these Wip1 mutants inhibits dephosphorylation of Thr-68 in Chk2 by Wip1 and anti-apoptotic function of Wip1. These results provide a molecular basis for developing novel anti-cancer drugs, targeting oncogenic Wip1 phosphatase.     

Expression of a Homeostatic Regulator, Wip1 (Wild-type p53-induced Phosphatase), Is Temporally Induced by c-Jun and p53 in Response to UV Irradiation

Wild-type p53-induced phosphatase (Wip1) is induced by p53 in response to stress, which results in the dephosphorylation of proteins (i.e. p38 MAPK, p53, and uracil DNA glycosylase) involved in DNA repair and cell cycle checkpoint pathways. p38 MAPK-p53 signaling is a unique way to induce Wip1 in response to stress. Here, we show that c-Jun directly binds to and activates the Wip1 promoter in response to UV irradiation. The binding of p53 to the promoter occurs earlier than that of c-Jun. In experiments, mutation of the p53 response element (p53RE) or c-Jun consensus sites reduced promoter activity in both non-stressed and stressed A549 cells. Overexpression of p53 significantly decreased Wip1 expression in HCT116 p53^+/+ cells but increased it in HCT116 p53^−/− cells. Adenovirus-mediated p53 overexpression greatly decreased JNK activity. Up-regulation of Wip1 via the p38 MAPK-p53 and JNK-c-Jun pathways is specific, as demonstrated by our findings that p38 MAPK and JNK inhibitors affected the expression of the Wip1 protein, whereas an ERK inhibitor did not. c-Jun activation occurred much more quickly, and to a greater extent, in A549-E6 cells than in A549 cells, with delayed but fully induced Wip1 expression. These data indicate that Wip1 is activated via both the JNK-c-Jun and p38 MAPK-p53 signaling pathways and that temporal induction of Wip1 depends largely on the balance between c-Jun and p53, which compete for JNK binding. Moreover, our results suggest that JNK-c-Jun-mediated Wip1 induction could serve as a major signaling pathway in human tumors in response to frequent p53 mutation.     

Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MAP kinase phosphatase-2 and their ability to activate the phosphatase catalytically

MKP-2 is a member of the mitogen-activated protein (MAP) kinase phosphatase family which has been suggested to play an important role in the feedback control of MAP kinase-mediated gene expression. Although MKP-2 preferentially inactivates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) MAP kinase subfamilies, the mechanisms underlying its own regulation remain unclear. In this report, we have examined the MKP-2 interaction with and catalytic activation by distinct MAP kinase subfamilies. We found that the catalytic activity of MKP-2 was enhanced dramatically by ERK and JNK but was affected only minimally by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, whereas JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the NH(2)-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised the catalytic activation of MKP-2 by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 whereas MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for the substrate preference of MKP-2 and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function.     

Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation

Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that switches off DNA damage checkpoint responses by the dephosphorylation of certain proteins (i.e. p38 mitogen-activated protein kinase, p53, checkpoint kinase 1, checkpoint kinase 2, and uracil DNA glycosylase) involved in DNA repair and the cell cycle checkpoint. Emerging data indicate that Wip1 is amplified or overexpressed in various human tumors, and its detection implies a poor prognosis. In this study, we show that Wip1 interacts with and dephosphorylates BAX to suppress BAX-mediated apoptosis in response to γ-irradiation in prostate cancer cells. Radiation-resistant LNCaP cells showed dramatic increases in Wip1 levels and impaired BAX movement to the mitochondria after γ-irradiation, and these effects were reverted by a Wip1 inhibitor. These results show that Wip1 directly interacts with and dephosphorylates BAX. Dephosphorylation occurs at threonines 172, 174 and 186, and BAX proteins with mutations at these sites fail to translocate efficiently to the mitochondria following cellular γ-irradiation. Overexpression of Wip1 and BAX, but not phosphatase-dead Wip1, in BAX-deficient cells strongly reduces apoptosis. Our results suggest that BAX dephosphorylation of Wip1 phosphatase is an important regulator of resistance to anticancer therapy. This study is the first to report the downregulation of BAX activity by a protein phosphatase.     

Mitogen-activated protein kinase (MAPK) phosphatase 3-mediated cross-talk between MAPKs ERK2 and p38alpha

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.     

USP7 controls Chk1 protein stability by direct deubiquitination

Chk1, an essential checkpoint kinase in the DNA damage response pathway (DDR), is tightly regulated by both ATR-dependent phosphorylation and proteasome-mediated degradation. Here we identify ubiquitin hydrolase USP7 as a novel regulator of Chk1 protein stability. USP7 was shown before to regulate other DDR proteins such as p53, Hdm2 and Claspin, an adaptor protein in the ATR-Chk1 pathway required for Chk1 activation. Depletion or inhibition of USP7 leads to lower Chk1 levels. The decreased Chk1 protein after USP7 knock down cannot be rescued by simultaneously elevating Claspin levels, demonstrating that the effect of USP7 on Chk1 is independent of its known effect on Claspin. Conversely, overexpression of USP7 wild type, but not a catalytic mutant version, elevates Chk1 levels and increases the half-life of Chk1 protein. Importantly, wild type, but not catalytic mutant USP7 can deubiquitinate Chk1 in vivo and in vitro, confirming that USP7 directly regulates Chk1 protein levels. Finally we show that USP7 catalytic mutant is (mono-)ubiquitinated, which suggests auto-deubiquitination by this ubiquitin hydrolase, possibly important for its regulation.     

Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein (SPRED1), a tyrosine-protein phosphatase non-receptor type 11 (SHP2) substrate in the Ras/extracellular signal-regulated kinase (ERK) pathway

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.