©La nd esBi o s ci en ce2004. N o tf or d i s t ri bu t i o n .[Cancer Biology &Therapy 3:2, 156-161, February 2004]; ©2004 Landes BioscienceGen Sheng WuCorrespondence to: Gen Sheng Wu; Program in Molecular Biology and Human Genetics; Karmanos Cancer Institute; Department of Pathology; Wayne State University School of Medicine; Room E216; The Prentis Building; 110 East Warren Avenue; Detroit, Michigan 48201 USA; Tel.: 313.833.0715x2328; Fax:313.831.7518; Email: wug@ Received 01/13/04; Accepted 01/13/04Previously published online as a Cancer Biology &Therapy E-publication:/journals/cbt/abstract.php?id=614KEY WORDSp53, MAPK , phosphatases, phosphorylation,transcriptional control, cell cycle, apoptosisACKNOWLEDGEMENTSI thank Drs. Larry H. Matherly and Ray Mattingly for critical reading of this manuscript.This work was supported by a grant from National Cancer Institute (1R01 CA100073-01 A1).I apologize to those colleagues whose work could not be cited due to space limitations.ReviewThe Functional Interactions Between the p53 and MAPK Signaling PathwaysABSTRACTThe p53 tumor suppressor protein exerts its growth inhibitory activity by activating and interacting with diverse signaling pathways. As a downstream target, p53 protein is phosphorylated and activated by a number of protein kinases in response to stressful stimuli.As an upstream activator, activated p53 acts as a transcription factor to induce and/or suppress a number of genes whose expression leads to the activation of diverse signaling pathways. p53 protein can also interact with a number of proteins, resulting in an increase or decrease in p53 activity itself. The activation of p53 leads to many outcomes in cells, including cell cycle arrest and apoptosis. It has become clear that the p53 protein can functionally interact with the mitogen-activated protein kinase (MAPK) pathways,including the stress-activated protein kinase [SAPK/c-Jun N-terminal protein kinase (JNK)],the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal related kinase (ERK). Upon exposure to stressful stimuli, MAP kinases phosphorylate and activate p53, leading to p53-mediated cellular responses. Recent studies have suggested a role of p53 as an upstream activator to regulate MAPK signaling via the transcriptional acti-vation of members of the dual specificity phosphatase family. Because both the p53 and MAPK signaling pathways are altered in the majority of human tumors, understanding their functional interaction may provide new insights into the deregulated cell proliferation and survival that is characteristic of cancer.INTRODUCTIONp53 is the most commonly mutated gene in human cancer. The p53 tumor suppressor protein is a nuclear phosphoprotein with a short half-life that is regulated mainly through post-translational modifications. Upon stressful stimuli, such as DNA damage and oncogene activation, p53 protein is modified through multiple post-translational events, including phosphorylation and acetylation.1-3These modifications stabilize and activate p53 protein.In addition, a recent study suggested a transcriptional regulation of p53 by the interferon signaling pathway.4Once p53 is activated, it acts as a transcription factor for many genes that contain the consensus p53-binding sites in their promoters or intronic sequences.5p53 can also suppress the expression of a number of genes whose expression may favor cell survival. It is well accepted that the transcriptional activity of p53 plays a major role in tumor suppression, although transcription-independent activity is also suggested by its growth inhibitory activity. Activation of p53 triggers a number of signaling pathways that lead to cell cycle arrest, apoptosis, senescence, DNA repair and antiagiogenesis.The MAP kinase signaling pathways mainly consists of three subfamilies; the stress-activated protein kinase [SAPK/c-Jun N-terminal protein kinase (JNK)], the p38mitogen-activated protein kinase (MAPK ), and the extracellular signal related kinase (ERK)6,7(Fig. 1). Members of the MAPK family can be induced by multiple stimuli including growth factors and stresses, leading to diverse cellular responses. In the presence of stimuli, these MAPK s are activated through the reversible phosphorylation of both threonine and tyrosine residues of the TXY motif in the catalytic domain by upstream dual-specificity kinases. These activating kinases are called MAPK kinases and include MKK1/2 (selective for ERK1/2 activation), MKK3/6 (selective for p38 activation) and MKK-4/7 (selective for JNK activation)8-10(Fig. 1). Once MAP kinases are activated, they function as effector protein kinases to phosphorylate a number of substrates including p53, leading to many changes in cells, such as cell growth, differentiation and apoptosis.9Since the p53 signaling pathways including its activation by upstream kinases, regulation of its downstream targets, and the biological consequences of its activation are complex,this review will focus on the interactions between the p53 and MAPK pathways, including©La nd esBi o s ci en ce2004. N o tf or d i s t ri bu t i o n .MAPK-mediated p53 phosphorylation and p53-mediated transcrip-tional inhibition of MAPK signaling.PHOSPHORYLATION AND ACTIVATION OF p53BY THE MAP KINASESHuman p53 is a 393 amino acid protein that consists of four functional domains: an N-terminal transactivation domain, a proline rich domain, a DNA binding domain, and a C-terminal domain with a tetramerization and a nuclear localization signaling sequence (Fig. 2). There are a number of protein kinases that can phosphorylate p53 at a number of sites, which mainly reside in either the N-ter-minus or C-terminus. One such kinase is the ataxiatelangiectasia mutated (ATM) protein, a member of the phosphoinositide-3kinase-related kinase (PI 3K) superfamily. Upon DNA damage, such as that resulting from ionizing radiation, ATM phosphorylates p53at several sites including serines 9, 15, 20, and 46 directly or indi-rectly.11-13In addition, ATR (ATM-Rad3-related), another member of this PI 3K superfamily, has been shown to phosphorylate p53 at both serines 15 and 37.14,15Furthermore, several additional kinases have been shown to phosphorylate p53 at various sites upon expo-sure to stressful stimuli, including DNA-PK, casein kinases I and II,CDK-activating kinase (CAK), cdc2, cdk2, and protein kinase c.16-21Interestingly, the MAP kinases including p38, JNKs and ERKs have been shown to phosphorylate p53 in response to different stressfulstimuli, and such phosphorylation can initiate the p53 response,leading to cell cycle arrest and apoptosis.p38.The p38 pathway is activated by stressful stimuli and cytokines. Stressful stimuli and cytokines can lead to activation of several upstream kinases including MLKs, TAK-1 and Ask-1.8These activated kinases then phosphorylate and activate downstream kinases MK K 3 and MK K 6. Once MK K 3 and MK K 6 are activated, they phosphorylate and activate p38.22,23Activated p38 then phosphory-lates a number of cellular substrates,leading to diverse cellular responses,such as apoptosis and cell survival.8A number of studies have shown that p38 plays an important role in activation of p53 via a mechanism involving phos-phorylation. In normal human ker-atinocytes, UVB activates p38, which then phosphorylates p53 on serine 15.This p38-dependent phosphorylation leads to p53 accumulation in the cyto-plasm and thus prevents p53 from entering into the nucleus; this provides a protective mechanism against UV-induced cell death.24Consistent with this role of p38 to regulate p53, p38 phosphorylates p53 at serines 15 and 20 in response toUVB radiation in a mouse JB6 epidermal cell line.25,26It has also been shown that Gadd45a, a p53 target, regulates the stabilization of p53 that is induced by UVB through p38 because this induc-tion is abrogated in the presence of a p38 kinase inhibitor.27Inhibition of p38 activation correlates with the dereg-ulation of p53 activation and, moreover,p38 forms a complex with Gadd45 proteins.28In rabbit articularchondrocytes, nitric oxide-induced apoptosis involves p38-mediated p53 activation, and the blockade of p38 kinase activity correlates with reduced p53 accumulation and caspase-3 activity.29A further study shows that nitric oxide-induced activation of p38 kinase phosphory-lates p53 at serine 15, which results in accumulation of p53 protein and subsequent induction of apoptosis.30Further, in human prostate cancer cell line, methoxyestradiol-induced apoptosis involves Figure 1. Schematic diagram showing three major MAPK signaling cascades and their inhibition by members of the dual specificity phosphatase family.These three major MAPK pathways are the extracellular-signal regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway,C-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK/MAPK) signaling pathway, and the p38/MAPK signaling pathway. All three pathways can be inactivated by members of the dual specificity phosphatase family including PAC1, MKP1 and DUSP5.Figure 2. A schematic diagram of the functional domains of p53 protein and its phosphorylation by MAP kinases. p53 protein consists of four functional domains: a transactivation domain (TA), a proline rich domain (PRD), a DNA binding domain (DBD), and a C-terminal region that contains a nuclear localization sequence (NLS) and a tetramerization domain (TETRA). Upon stressful stimuli, MAP kinases are activated and in turn, phosphorylate and activate p53, leading to p53-mediated cellular responses. Since MAPK-medi-ated p53 phosphorylation at certain residues is observed differently between mouse and human cells, thefollowing phosphorylated sites that occur either in human (h) or mouse (m) cells or both are indicated: p38can phosphorylate p53 at ser 15 (h and m), ser 20 (m), ser 33 (h and m), ser 37 and 46 (h and m), and ser 389 (m); JNKs can phosphorylate p53 at ser 15 (m), ser 20 (m), ser 34 (m) and thr 81 (h); and ERKs can phosphorylate p53 at ser 15 (h and m), thr 73 (m) and thr 83 (m). ser: serine; thr: threonine.©La nd esBi o s ci en ce2004. N o tf or d i s t ri bu t i o n .p53 activation through p38/JNK -dependent NF-κB/AP-1.31In neurons, p38-mediated p53 phosphorylation at serine 15 and subse-quent p53 induction is responsible for hypoxic neuronal death.32In addition to its phosphorylation of p53 at serines 15 and 20,p38 has been shown to phosphorylate p53 at several additional sites including serines 33, 37 and 46. For example, upon osmotic shock,p38 is activated and then phosphorylates p53 at serine 33 and subsequently causes a G1 arrest.33Interestingly, p38 is also involved in phosphorylating p53 at serine 33 in anticancer-drug-induced apoptosis.34p38 phosphorylates p53 at serines 33 and 46 in-vitro and inhibition of p38 activity during UV irradiation decreases phosphorylation of p53 at serines 15, 33 and 37 and reduces UV-induced p53-dependent apoptosis.35Finally, p38 has been shown to phosphorylate p53 at serine 389 in response to UV radia-tion.36Therefore, stress-induced p38 acts as a protein kinase to phosphorylate p53 at various serine residues, which, in turn, activatesp53, leading to p53-mediated cellular responses, such as the induction of apoptosis.JNKs.The stress-activated protein kinase (SAPK) pathway is alsocalled the Jun N-terminal kinase pathway as the kinase could phos-phorylate the NH2-terminus of the transcription factor c-JUN.37,38Like p38, JNK activation involves a phosphorylation cascade. Upon stressful stimuli, MEK K 1 is activated and subsequently activates MK K 4 and MK K 7.8Activated MK K 4/7 then phosphorylate and activate JNKs.39-41Once JNKs are activated, they can phosphorylate a number of substrates, leading to activation of many signaling pathways,8 including the p53 pathway.The main role of JNKs in regulating the p53 pathway is currently thought to be through their ability to phosphorylate p53 protein.Upon UVB radiation, JNKs are directly involved in phosphoryla-tion of p53 at serine 20, resulting in an increase in p53-dependent transcriptional activity. This response is suppressed in JNK1 or JNK2 knockout cells.26Under oxidative stress,JNKs can phosphorylate p53 at serine 15.42In addition,JNK phosphorylates p53 at threonine 81 in response to DNA damage and stress-inducing agents, and substitu-tion of this site (T81A) attenuates UV-and JNK-medi-ated p53 stabilization and subsequently impairs p53-mediated G1 arrest.43A further study has identified the amino-terminus of JNK as a functional inducer that activates p53 transcriptional activity.44In addition to phosphorylation of p53 upon DNA damage, JNK has been suggested to regulate p53 stability in nonstressed cells through an MDM2-independent mechanism.45Consistently, MEKK1, the upstream regulator of JNK,has been shown to regulate p53 stability.46Finally, it has been shown that JNK1 can phosphorylate p53 at serine 34 in murine cells.47Thus, like p38, JNKs function as upstream kinases to phosphorylate p53 at various sites in response to DNA damage and subsequently stabilize and activate p53 transcriptional activity.ERK1/2.Unlike p38 and JNK, ERK is generally acti-vated by growth actors (Fig. 1). Upon growth factor stimuli, the Ras/Raf pathway is activated and subse-quently activates MEK 1/2 kinases.48,49ActivatedMEK1/2 then activate ERK 1/2,50leading to activation of a number of transcription factors.8In spite of the fact that activation of p53 by growth factors has not been clearly established, phosphorylation of p53 by ERKs has been well observed in a number of experimental sys-tems. In ovarian cells, ERK1/2 have been shown to berequired for p53 phosphorylation at serine 15 and apoptosis inducedby cisplatin.51ERK also regulates p53 activation and subsequent cellcycle arrest induced by colcemid, and the selective MEK1 inhibitor PD098059 attenuates colcemid-induced p53 activation and G 1cell cycle arrest.52Furthermore, ERKs mediate resveratrol-induced acti-vation of p53 and apoptosis through phosphorylation of p53 at ser-ine 15.53Interestingly, in the murine system, ERK phosphorylates p53 at threonines 73 and 83, a proline-rich region that contains a transactivation domain of p53.54Since some of phosphorylated sites on p53 (e.g., thr 73 and 83) are detected only in mouse cells but not in human cells, it is believed that there is differential regulation of p53 phosphorylation between mouse and human cells.55Thus,ERKs act as upstream activators to phosphorylate p53 at serine and threonine residues, leading to p53 activation and subsequent cellular responses.REGULATION OF MAPK SIGNALING BY p53VIA A TRANSCRIPTIONAL MECHANISMIt is well known that the transcriptional activity of p53 plays a pivotal role in its tumor suppression activity.2There is a growing list of the genes that are transcriptionally regulated by p53, and activa-tion of these targets leads to distinct cellular responses in cells.3For example, p53-mediated growth arrest involves the activation of p21,56,57Gadd45,58and 14-3-3σ.59p53-induced apoptosis, on the other hand, involves the activation of several genes, which includeBax,60,61Fas/APO1,62KILLER/DR5,63,64Pigs,65IGF-BP3,66PERP ,67Noxa,68p53AIP1,69p53DINP1,70PUMA,71,72Bid 73and caspase 6.74It has also been shown that p53 can induce antiangio-genesis by upregulating Tsp1,75BaI1 and GD-AiF .76,77In addition,p53 induces MDM2, an oncogene that negatively regulates p53Figure 3. Models for the roles of phosphatases in p53-mediated cellular responses. DNAdamage activates p38 and, in turn, activates p53 via phosphorylation. Activated p53 can transcriptionally induce a number of genes, leading to apoptosis, which may be negatively regulated by p53-mediated induction of Wip1 because Wip1 can dephosphorylate p38and thus suppress its role in phosphorylating p53. It has been shown that DNA damage such as oxidative stress activates p53, leading to upregulation of PAC1 and DUSP5.Because ERK1/2 are believed to play a negative role in apoptosis in some cell types, inac-tivation of ERK1/2 by p53-mediated PAC1 and DUSP5 leads to induction of apoptosis. In the case of MKP1, activation of MKP1 can inhibit mitogen-mediated ERK activation and subsequently block re-entry of cells into the cell cycle. Therefore, it is believed that p53-mediated MKP1 induction controls re-entry of arrested cells into the cell cycle in response to mitogens in the absence of DNA damage, although it is not clear how p53 is activated in response to growth factor stimuli.©La nd esBi o s ci en ce2004. N o tf or d i s t ri bu t i o n .stabilization.78,79Interestingly, recent investigations have identified p53-regulated transcription of four phosphatases that are known to negatively regulate MAPK signaling: Wip1, MK P1, PAC1 and DUSP580-83(Fig. 3).Wip1.Wip1 was the first phosphatase to be identified as a tran-scriptional target of p53. Wip1 is a serine/threonine phosphatase belonging to the type 2 class that are insensitive to phosphatase inhibitors such as okadaic acid.80Wip1 is induced by ionizing radiation in a p53-dependent manner.80Induction of Wip1 by p53leads to p53-mediated G 1arrest in response to DNA damage.80A mechanistic study has revealed that Wip1 can inactivate p38 activity in response to UV radiation.84p38 can phosphorylate p53 at serines 33 and 46,35and p53-dependent induction of Wip1 could dephos-phorylate p38 and, in turn, downregulate UV-induced p53 phospho-rylation at these residues.35This study suggests that Wip1 may serve as a negative feedback loop to control the MAPK-p53 signaling path-way. Consistent with a role for Wip1 in the p53 pathway, fibroblasts derived from Wip1 knockout mice exhibit a premature senescence and a defect in entering mitosis.85MKP1.MKP1, also known as CL100, 3CH134, and Erp, is the founding member of the dual-specificity protein phosphatase fami-ly 86-88and found to be a new target of p53.82This family includes MKP1, MKP2, MKP3, MKP4, MKP5, VHR, Pac1, hVH2, hVH3,Pyst1 and Pyst2.89Because phosphorylation is required for the activation of MAP kinases, dephosphorylation by members of this family inactivates the MAP kinases and thus inhibits their signaling 90(Fig. 1). MKP1 was originally identified as an ERK-specific phos-phatase.88,91It is now known that MKP1 can also inhibit JNK and p38.92,93Constitutive MK P1 expression blocks G 1specific gene expression,94suggesting a role in the cell cycle. We have shown that MK P1 is induced in cells when p53 is conditionally induced or when cells are infected with p53 adenoviruses.82p53 regulates MKP1 induction through a p53-responsive element residing in the second intron of this gene.93Interestingly, overexpression of MKP1impairs re-entry of arrested cells into the cell cycle in response to growth factor stimuli.93This regulation appears to be DNA damage independent since p53 is generally induced and activated following DNA damage. Furthermore, inhibition of phosphatase activity by vanadate can partially relieve p53-mediated G 1arrest.93These data indicate that p53 may negatively control re-entry of arrested cells into the cell cycle in response to mitogens in the absence of DNA damage (Fig. 3).PAC1.Pac1 (phosphatase activated in cells 1), also known as DUSP2 (dual specificity phosphatase 2), is another member of the dual-specificity protein phosphatase family capable of inactivating all three major MAPK signaling pathways 89(Fig. 1). Using microarray technology, Pac1 was isolated as a p53 target.81Unlike other p53targets such as p21, Pac1 induction requires both functional p53 and serum starvation/oxidative damage.81Overexpression of Pac1 sensitizes cells to apoptosis induced by serum starvation and H 202treatment.Importantly, reduction of Pac1 by siRNA approach renders cells resistant to oxidative damage-induced apoptosis.81These data suggest that Pac1 may be involved in oxidative damage-induced p53-mediated apoptosis. Interestingly, p53 transcriptionally regulates Pac1 expression through a novel binding site residing in the promoter of the Pac1gene.81This binding site is distinct from the well-described p53consensus sequence.5Because Pac1 is able to inactivate the ERK 1/2-mediated MAPK pathway, and because p53 can induce Pac1 induction in response to oxidative stress, it is believed that p53-mediated apoptosis induced by oxidative stress is through Pac1-dependent inactivation of ERK pathways (Fig. 3).DUSP5.DUSP5 is the third member of the dual-specificity protein phosphatase family that has been shown to inhibit all three major MAPK pathways with preferential specificity 89(Fig. 1) and that has been recently identified as the new target for p53.83DUSP5is induced in cells by p53 overexpression.83Induction of DUSP5 by p53 appears to be mediated through a p53-binding site in the pro-moter of the DUSP5 gene.83Interestingly, a functional study sug-gested DUSP5 can preferentially dephosphorylate ERK s,83thus providing another link between the p53 and MAPK signaling pathways (Fig. 3).p53 REGULATES THE MAPK PATHWAYS AS A PROTECTIVE MECHANISM AGAINST CELLULAR INSULTSIn addition to phosphatases, there are several other genes that can functionally link the p53 to MAPK pathways. Using a p53-inducible cell system, Lee et al. found that inducible expression of p53 in human bladder cancer cells leads to an activation of MEK and its downstream kinase ERK, but not p38.95Activation of ERK by p53 requires transcriptionally active p53 because p53 mutants that lack transcriptional activity fail to do so.95This suggests that p53 can transcriptionally activate ERK/MAPK signaling. A mecha-nistic study of p53-mediated MAPK activation has led to the identi-fication of heparin-binding epidermal growth factor-like growth factor (HB-EGF) as a new p53 target.95,96Through binding to the EGF receptor, HB-EGF can activate the phosphatidylinositol 3-kinase (PI3K )/AK T pathway, a major cell survival pathway.Inhibition of HB-EGF activity can decrease p53-mediated MAPK activation, while overexpression of HB-EGF protects cells from oxidative damage-induced apoptosis.96These studies suggest that besides activation of the signaling pathways leading to growth arrest and apoptosis, p53 can also activate survival pathways in order to maintain the balance between life and death in response to DNA damage. This idea is supported by discovery of the involvement of discoidin domain receptor 1 (DDR1) in the p53 pathway.DDR1 was initially identified as a member of the receptor tyrosine kinase family. DDR1 was induced and tyrosine phosphorylated fol-lowing genotoxic stress in a p53-dependent manner.97Since blocking DDR1 expression by a means of dominant negative mutants or antisense constructs can sensitize cells to p53-mediated apoptosis induced by DNA damage, it is likely that DDR1 serves as another negative regulator that controls p53 responses via activation of the ERK pathway. These data are consistent with the role of the Ras/Raf/MAPK cascade in regulating p53-mediated cellular responses.98,99A recent study has identified cyclooxygense-2 (Cox2) as the third gene that can serve as a downstream effector to participate in the p53-MAPK pathway.100Cox2 is a key enzyme in the synthesis of prostaglandins and thromboxanes. Cox2 is highly induced in response to growth factors. It is known that the ERK/MAPK signal can activate Cox2 expression, that activation of Cox2 leads to cell survival, and that Cox2 is induced in a p53-dependent manner.100Interestingly, induction of Cox2 by p53 is not due to binding of p53to a p53 responsive element in the Cox2 genomic sequence, but rather by p53-induced activation of ERK/MAPK.100Like HB-EGF and DDR1, activation of Cox2 can inhibit p53-mediated apoptosis and chemosensitivity in response to genotoxic stress.100T ogether,these investigations have suggested that the outcomes of p53 activation are dependent on the balance between activation of death-related genes and survival-related genes.©La nd esBi o s ci en ce2004. N o tf or d i s t ri bu t i o n .CONCLUSIONSMuch of our understanding of the functional interactions between the p53-MAPK pathways has dealt with the role of MAP kinases in phosphorylating and activating p53 in response to DNA damage. Recent findings of the MAPK inhibitory phosphatases as p53 targets have provided a more complex and comprehensive picture of how p53 functionally interacts with the MAPK signaling pathways. On the one hand, MAPK-dependent p53 phosphoryla-tion following DNA damage can lead to p53-dependent cellular responses such as cell cycle arrest and apoptosis. On the other hand,activation of phosphatases by p53 suggests a mechanism by which p53 negatively regulates MAPK -dependent signaling. Although activation of p53, in most cases, causes growth arrest and apoptosis,identification of p53 target genes (e.g., DDR1) that are stimulatory components of the MAPK pathways suggests a role for p53 in cell survival. It is well known that the outcomes of p53 activation by stressful stimuli depend on many factors such as cell context and type and duration of stresses. This pattern of complex and integrated responses is also true in the case of the activation of the MAPK pathways. 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