p38γ MAPK delays myelination and remyelination and is abundant in multiple sclerosis lesions.

Multiple sclerosis is a chronic inflammatory disease in which disability results from the disruption of myelin and axons. During the initial stages of the disease, injured myelin is replaced by mature myelinating oligodendrocytes that differentiate from oligodendrocyte precursor cells. However, myelin repair fails in secondary and chronic progressive stages of the disease and with ageing, as the environment becomes progressively more hostile. This may be attributable to inhibitory molecules in the multiple sclerosis environment including activation of the p38MAPK family of kinases. We explored oligodendrocyte precursor cell differentiation and myelin repair using animals with conditional ablation of p38MAPKγ from oligodendrocyte precursors. We found that p38γMAPK ablation accelerated oligodendrocyte precursor cell differentiation and myelination. This resulted in an increase in both the total number of oligodendrocytes and the migration of progenitors ex vivo and faster remyelination in the cuprizone model of demyelination/remyelination. Consistent with its role as an inhibitor of myelination, p38γMAPK was significantly downregulated as oligodendrocyte precursor cells matured into oligodendrocytes. Notably, p38γMAPK was enriched in multiple sclerosis lesions from patients. Oligodendrocyte progenitors expressed high levels of p38γMAPK in areas of failed remyelination but did not express detectable levels of p38γMAPK in areas where remyelination was apparent. Our data suggest that p38γ could be targeted to improve myelin repair in multiple sclerosis.

p38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA.

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Molecular characterization reveals that OsSAPK3 improves drought tolerance and grain yield in rice.

BACKGROUND: Many data suggest that the sucrose non-fermenting 1-related kinases 2 (SnRK2s) are very important to abiotic stress for plants. In rice, these kinases are known as osmotic stress/ABA-activated protein kinases (SAPKs). Osmotic stress/ABA-activated protein kinase 3 (OsSAPK3) is a member of SnRK2II in rice, but its function is still unclear. RESULTS: The expression of OsSAPK3 was up regulated by drought, NaCl, PEG and ABA. OsSAPK3 mutated seedings (sapk3-1 and sapk3-2) showed reduced hypersensitivity to exogenous ABA. In addition, under drought conditions, sapk3-1 and sapk3-2 showed more intolerance to drought, including decreased survival rate, increased water loss rate, increased stomatal conductance and significantly decreased expression levels of SLAC1 and SLAC7. Physiological and metabolic analyses showed that OsSAPK3 might play an important role in drought stress signaling pathway by affecting osmotic adjustment and osmolytes, ROS detoxification and expression of ABA dependent and independent dehydration-responsive genes. All gronomic traits analyses demonstrated that OsSAPK3 could improve rice yield by affecting the regulation of tiller numbers and grain size. CONCLUSION: OsSAPK3 plays an important role in both ABA-dependent and ABA-independent drought stress responses. More interestingly, OsSAPK3 could improve rice yield by indirectly regulating tiller number and grain size. These findings provide new insight for the development of drought-resistant rice.

Identification of Potential p38γ Inhibitors via In Silico Screening, In Vitro Bioassay and Molecular Dynamics Simulation Studies.

Protein kinase p38γ is an attractive target against cancer because it plays a pivotal role in cancer cell proliferation by phosphorylating the retinoblastoma tumour suppressor protein. Therefore, inhibition of p38γ with active small molecules represents an attractive alternative for developing anti-cancer drugs. In this work, we present a rigorous and systematic virtual screening framework to identify potential p38γ inhibitors against cancer. We combined the use of machine learning-based quantitative structure activity relationship modelling with conventional computer-aided drug discovery techniques, namely molecular docking and ligand-based methods, to identify potential p38γ inhibitors. The hit compounds were filtered using negative design techniques and then assessed for their binding stability with p38γ through molecular dynamics simulations. To this end, we identified a promising compound that inhibits p38γ activity at nanomolar concentrations and hepatocellular carcinoma cell growth in vitro in the low micromolar range. This hit compound could serve as a potential scaffold for further development of a potent p38γ inhibitor against cancer.

p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration.

Non-alcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38γ and p38δ) have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of obese patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to diet-induced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential target for NAFLD therapy.

p38δ MAPK regulates aggresome biogenesis by phosphorylating SQSTM1 in response to proteasomal stress.

Aggresome formation is a major strategy to enable cells to cope with proteasomal stress. Misfolded proteins are assembled into micro-aggregates and transported to the microtubule organizing center (MTOC) to form perinuclear aggresomes before their degradation through autophagy. So far, multiple factors have been identified as the activators of micro-aggregate formation, but much less is known about the regulatory mechanisms of their transport. Here, we report that proteasomal stress leads to the activation of p38 MAPK family members. Two of them, p38γ (MAPK12) and p38δ (MAPK13), are dispensable for micro-aggregate formation but are required for their targeting to the MTOC. Interestingly, p38δ promotes micro-aggregate transport by phosphorylating SQSTM1, a major scaffold protein that assembles soluble ubiquitylated proteins into micro-aggregates. Expression of the phospho-mimetic mutant of SQSTM1 in p38δ-knockout cells completely rescued their aggresome formation defects and enhanced their resistance to proteasomal stress to wild-type levels. This study reveals p38δ-mediated SQSTM1 phosphorylation as a critical signal for the targeting of micro-aggregates to the MTOC and provides direct evidence for the survival advantages associated with aggresome formation in cells under proteasomal stress.

p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice.-Loonat, A. A., Martin, E. D., Sarafraz-Shekary, N., Tilgner, K., Hertz, N. T., Levin, R., Shokat, K. M., Burlingame, A. L., Arabacilar, P., Uddin, S., Thomas, M., Marber, M. S., Clark, J. E. p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

A Dynamic Switch in Inactive p38γ Leads to an Excited State on the Pathway to an Active Kinase.

The inactive state of mitogen-activated protein kinases (MAPKs) adopts an open conformation while the active state exists in a compact form stabilized by phosphorylation. In the active state, eukaryotic kinases undergo breathing motions related to substrate binding and product release that have not previously been detected in the inactive state. However, docking interactions of partner proteins with inactive MAPK kinases exhibit allostery in binding of activating kinases. Interactions at a site distant from the activation loop are coupled to the configuration of the activation loop, suggesting that the inactive state may also undergo concerted dynamics. X-ray crystallographic studies of nonphosphorylated, inactive p38γ reveal differences in domain orientations and active site structure in the two molecules in the asymmetric unit. One molecule resembles an inactive kinase with an open active site. The second molecule has a rotation of the N-lobe that leads to partial compaction of the active site, resulting in a conformation that is intermediate between the inactive open state and the fully closed state of the activated kinase. Although the compact state of apo p38γ displays several of the features of the activated enzyme, it remains catalytically inert. In solution, the kinase fluctuates on a millisecond time scale between the open ground state and a weakly populated excited state that is similar in structure to the compact state observed in the crystal. The nuclear magnetic resonance and crystal structure data imply that interconversion between the open and compact states involves a molecular switch associated with the DFG loop.

p38gamma overexpression promotes renal cell carcinoma cell growth, proliferation and migration.

Recent studies have proposed that p38gamma (p38γ) might be critically involved in tumorigenesis and cancer progression. Its expression and potential functions in human renal cell carcinoma (RCC) are studied here. We show that p38γ mRNA and protein levels are upregulated in human RCC tissues, as compared to its levels in the surrounding normal renal tissues. p38γ upregulation was also detected in established (786-O line) and primary human RCC cells. Functional studies in 786-O cells and primary human RCC cells demonstrated that p38γ silencing (by targeted shRNAs) or CRISPR/Cas9-mediated p38γ knockout (KO) potently inhibited cell growth, viability, proliferation and migration. Furthermore, p38γ shRNA or KO in RCC cells decreased retinoblastoma (Rb) phosphorylation and downregulated cyclin E1/A expression. Additionally, significant apoptosis activation was detected in p38γ-silenced and p38γ-KO RCC cells. Contrarily, ectopic overexpression of p38γ facilitated cell growth, viability, proliferation and migration in RCC cells. Taken together, we show that p38γ overexpression promotes RCC cell growth, proliferation and migration. p38γ could be a novel therapeutic target for human RCC.

The K-Ras effector p38γ MAPK confers intrinsic resistance to tyrosine kinase inhibitors by stimulating EGFR transcription and EGFR dephosphorylation.

Mutations in K-Ras and epidermal growth factor receptor (EGFR) are mutually exclusive, but it is not known how K-Ras activation inactivates EGFR, leading to resistance of cancer cells to anti-EGFR therapy. Here, we report that the K-Ras effector p38γ MAPK confers intrinsic resistance to small molecular tyrosine kinase inhibitors (TKIs) by concurrently stimulating EGFR gene transcription and protein dephosphorylation. We found that p38γ increases EGFR transcription by c-Jun-mediated promoter binding and stimulates EGFR dephosphorylation via activation of protein-tyrosine phosphatase H1 (PTPH1). Silencing the p38γ/c-Jun/PTPH1 signaling network increased sensitivities to TKIs in K-Ras mutant cells in which EGFR knockdown inhibited growth. Similar results were obtained with the p38γ-specific pharmacological inhibitor pirfenidone. These results indicate that in K-Ras mutant cancers, EGFR activity is regulated by the p38γ/c-Jun/PTPH1 signaling network, whose disruption may be a novel strategy to restore the sensitivity to TKIs.

Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

ErbB4 Activated p38γ MAPK Isoform Mediates Early Cardiogenesis Through NKx2.5 in Human Pluripotent Stem Cells.

Activation of ErbB4 receptor signaling is instrumental in heart development, lack of which results in embryonic lethality. However, mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells, we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38γ MAPK isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38γ/NKx2.5 downregulation is followed by p38α/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38γ MAPK similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38γ signaling hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38γ to the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development.

Exenatide Regulates Substrate Preferences Through the p38γ MAPK Pathway After Ischaemia/Reperfusion Injury in a Rat Heart.

BACKGROUND: It is reported that glucagon-like peptide-1 (GLP-1) has direct cardioprotective effects. We hypothesise that Exenatide, a long half-life analog of GLP-1, might protect the heart against ischaemia/reperfusion (I/R) injury. In this study, the role and mechanism of Exenatide in I/R was investigated. METHODS: Two p38 mitogen-activated protein kinase (MAPK) isoforms p38α or p38γ, were knocked down by recombinant adeno-associated virus (rAAV) in male Sprague-Dawley rats. Then, rats were randomly treated with Exenatide or phosphate buffered saline (PBS) before I/R. Left ventricular function was measured. The translocation of glucose transporter 4 (GLUT4), GLUT1 and fatty acid transporter (FAT)/CD36 was assessed. RESULTS: Exenatide treatment increased the p38γ expression, but not p38α, in I/R rats. Exenatide significantly improved post-ischaemic cardiac function of I/R rats. The administration of Exenatide stimulated the translocation of GLUT4 and GLUT1, while it also increased the GLUT1 expression in the cytoplasm. Meanwhile, it reduced the translocation of FAT/CD36 (p<0.05). However, cardiac down-regulation of p38γ mediated by rAAV abolished not only the Exenatide-induced cardioprotective effects but also the GLUT4, GLUT1 and FAT/CD36 translocation. CONCLUSIONS: These results demonstrated that Exenatide improved cardiac function, increased translocation of GLUTs, and suppressed translocation of FAT/CD36 after myocardial I/R injury. This protective effect was mediated, at least in part, through modulation of the cardiac p38γ MAPK.

ErbB2 and p38γ MAPK mediate alcohol-induced increase in breast cancer stem cells and metastasis.

BACKGROUND: Both epidemiological and experimental studies suggest that excessive alcohol exposure increases the risk for breast cancer and enhances metastasis/recurrence. We have previously demonstrated that alcohol enhanced the migration/invasion of breast cancer cells and cancer cells overexpressing ErbB2/HER2 were more sensitive to alcohol exposure. However, the underlying mechanisms remain unclear. This study was designed to investigate the mechanisms underlying alcohol-enhanced aggressiveness of breast cancer. Cancer stem cells (CSCs) play a critical role in cancer metastasis and recurrence. METHODS: We evaluated the effect of chronic alcohol exposure on mammary tumor development/metastasis in MMTV-neu transgenic mice and investigated the cell signaling in response to alcohol exposure in breast cancer cells overexpressing ErbB2/HER2. RESULTS AND DISCUSSION: Chronic alcohol exposure increased breast cancer stem cell-like CSC population and enhanced the lung and colon metastasis in MMTV-neu transgenic mice. Alcohol exposure caused a drastic increase in CSC population and mammosphere formation in breast cancer cells overexpressing ErbB2/HER2. Alcohol exposure stimulated the phosphorylation of p38γ MAPK (p-p38γ) which was co-localized with phosphorylated ErbB2 and CSCs in the mammary tumor tissues. In vitro results confirmed that alcohol activated ErbB2/HER2 and selectively increased p-p38γ MAPK as well as the interaction between p38γ MAPK and its substrate, SAP97. However, alcohol did not affect the expression/phosphorylation of p38α/ß MAPKs. In breast cancer cell lines, high expression of ErbB2 and p-p38γ MAPK was generally correlated with more CSC population. Blocking ErbB2 signaling abolished heregulin ß1- and alcohol-stimulated p-p38γ MAPK and its association with SAP97. More importantly, p38γ MAPK siRNA significantly inhibited an alcohol-induced increase in CSC population, mammosphere formation and migration/invasion of breast cancer cells overexpressing ErbB2. CONCLUSIONS: p38γ MAPK is downstream of ErbB2 and plays an important role in alcohol-enhanced aggressiveness of breast cancer. Therefore, in addition to ErbB2/HER2, p38γ MAPK may be a potential target for the treatment of alcohol-enhanced cancer aggressiveness.

p38γ MAPK Is a Therapeutic Target for Triple-Negative Breast Cancer by Stimulation of Cancer Stem-Like Cell Expansion.

Triple-negative breast cancer (TNBC) is highly progressive and lacks established therapeutic targets. p38γ mitogen-activated protein kinase (MAPK) (gene name: MAPK12) is overexpressed in TNBC but how overexpressed p38γ contributes to TNBC remains unknown. Here, we show that p38γ activation promotes TNBC development and progression by stimulating cancer stem-like cell (CSC) expansion and may serve as a novel therapeutic target. p38γ silencing in TNBC cells reduces mammosphere formation and decreases expression levels of CSC drivers including Nanog, Oct3/4, and Sox2. Moreover, p38γ MAPK-forced expression alone is sufficient to stimulate CSC expansion and to induce epithelial cell transformation in vitro and in vivo. Furthermore, p38γ depends on its activity to stimulate CSC expansion and breast cancer progression, indicating a therapeutic opportunity by application of its pharmacological inhibitor. Indeed, the non-toxic p38γ specific pharmacological inhibitor pirfenidone selectively inhibits TNBC growth in vitro and/or in vivo and significantly decreases the CSC population. Mechanistically, p38γ stimulates Nanog transcription through c-Jun/AP-1 via a multi-protein complex formation. These results together demonstrate that p38γ can drive TNBC development and progression and may be a novel therapeutic target for TNBC by stimulating CSC expansion. Inhibiting p38γ activity with pirfenidone may be a novel strategy for the treatment of TNBC.

p38γ Mitogen-activated protein kinase signals through phosphorylating its phosphatase PTPH1 in regulating ras protein oncogenesis and stress response.

Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.

Delayed cell cycle progression in selenoprotein W-depleted cells is regulated by a mitogen-activated protein kinase kinase 4-p38/c-Jun NH2-terminal kinase-p53 pathway.

Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21(Cip1)-dependent G(1)-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G(1) arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G(1) arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G(1) arrest.

p38γ mitogen-activated protein kinase (MAPK) confers breast cancer hormone sensitivity by switching estrogen receptor (ER) signaling from classical to nonclassical pathway via stimulating ER phosphorylation and c-Jun transcription.

Estrogen receptor (ER) α promotes breast cancer growth by regulating gene expression through classical estrogen response element (ERE) binding and nonclassical (interaction with c-Jun at AP-1 sites) pathways. ER is the target for anti-estrogens such as tamoxifen (TAM). However, the potential for classical versus nonclassical ER signaling to influence hormone sensitivity is not known. Moreover, anti-estrogens frequently activate several signaling cascades besides the target ER, and the implications of these "off-target" signaling events have not been explored. Here, we report that p38γ MAPK is selectively activated by treatment with TAM. This results in both phosphorylation of ER at Ser-118 and stimulation of c-Jun transcription, thus switching ER signaling from the classical to the nonclassical pathway leading to increased hormone sensitivity. Unexpectedly, phosphorylation at Ser-118 is required for ER to bind both p38γ and c-Jun, thereby promoting ER relocation from ERE to AP-1 promoter sites. Thus, ER/Ser-118 phosphorylation serves as a central mechanism by which p38γ regulates signaling transduction of ER with its inhibitor TAM.