Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38γ mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38γ, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38γ phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Rasdependent malignant growth in vitro and/or in mice by a mechanism depending on its p38γ-binding activity. Moreover, Ras increases both p38γ and PTPH1 protein expression and there is a coupling of increased p38γ and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38γ/PTPH1 complex may be a novel target for Ras-dependent malignancies.

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38␥ MAPK, and the activated c-Jun then recruits p38␥ as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38␥ that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38␥ interacting with c-Jun. p38␥ requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38␥ are required for the trans-activation of MMP9. The active p38␥/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38␥ and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response. MAPKs3 (including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38s) are critical signaling cascades that convert upstream signals into biological responses such as cell proliferation, invasion, and transformation (1). MAPKs are believed to do so by phosphorylating and activating a group of transcription factors, which through binding regulatory DNA elements lead to altered gene transcription. c-Jun is a major component of the AP-1 transcription factor downstream of MAPKs, whereas AP-1 is composed of homodimers of the Jun family or its heterodimers with another transcription factor such as c-Fos to bind the consensus DNA elements TGAg/cTCA (2). c-Jun is activated by JNK through phosphorylation at Ser-63, Ser-73, Thr-91, and Thr-93, and by ERK and p38 via increased gene expression. Activated c-Jun/ AP-1 leads to a cell type-specific biological response through integrated gene expression (1). However, the exact mechanism by which c-Jun converts a MAPK activity into a target gene expression remains mostly unknown.p38 MAPKs consist of four family members (␣, ␤, ␥, and ␦) in which p38␣ is ubiquitously present, whereas p38␥ is highly expressed in certain cancers (3). In addition to well established regulatory effects in cytokine signaling and stress response, substantial evidence suggests that the p38␣ pathway functions as a tumor suppressor (4 -8). p38␥, on the other hand, is a 43-kDa protein with an unique C-terminal motif, KETXL, that can dock with the PDZ (PSD-95/Dlg/ZO-1 homology) domain of other proteins (9, 10). In contrast to p38␣, our recent studies showed that p38␥ is induced by Ras and required for Ras transformation and invasion (11, 12), indicating its oncogenic activity. The underlying mechanisms for p38␥ involvement in Ras tumorigenesis, however, have not been established. In this report, we show that p38␥ acts both as an activator and a cofactor for c-Jun in trans-activating MMP9, a critical matrix metalloprote...

Oxidative stress suppresses host immunity by generating oxidized lipid agonists of the platelet-activating factor receptor (PAF-R). Because many classical chemotherapeutic drugs induce reactive oxygen species (ROS), we investigated whether these drugs might subvert host immunity by activating PAF-R. Here we show that PAF-R agonists are produced in melanoma cells by chemotherapy that is administered in vitro, in vivo or in human subjects. Structural characterization of the PAF-R agonists induced revealed multiple oxidized glycerophosphocholines that are generated non-enzymatically. In a murine model of melanoma, chemotherapeutic administration could augment tumor growth by a PAF-R-dependent process that could be blocked by treatment with antioxidants or cyclooxygenase-2 inhibitors or by depletion of regulatory T cells. Our findings reveal how PAF-R agonists induced by chemotherapy treatment can promote treatment failure. Further, they offer new insights into how to improve the efficacy of chemotherapy by blocking its heretofore unknown impact on PAF-R activation.

Ras is believed to stimulate invasion and growth by different effector pathways, and yet, the existence of such effectors under physiologic conditions has not been shown. Estrogen receptor (ER), on the other hand, is both anti-invasive and proliferative in human breast cancer, with mechanisms for these paradoxical actions remaining largely unknown. Our previous work showed an essential role of p38; mitogenactivated protein kinase in Ras transformation in rat intestinal epithelial cells, and here, we show that p38; integrates invasive antagonism between Ras and ER to increase human breast cancer invasion without affecting their proliferative activity. Ras positively regulates p38; expression, and p38; in turn mediates Ras nonmitogenic signaling to increase invasion. Expression of the Ras/p38; axis, however, is trans-suppressed by ER that inhibits invasion and stimulates growth also by distinct mechanisms. Analysis of ER and its cytoplasmic localized mutant reveals that ER additionally binds to p38; protein, leading to its specific down-regulation in the nuclear compartment. A p38;-antagonistic activity of ER was further shown in a panel of breast cancer cell lines and was shown independent of estrogens by both ER depletion and ER expression. These results revealed that both Ras and ER use distinct pathways to regulate breast cancer growth and invasion, and that p38; specifically integrates their antagonistic activity to stimulate cell invasion. Selective targeting of p38;-dependent invasion pathways may be a novel strategy to control breast cancer progression. (Cancer Res 2006; 66(15): 7540-7)

Abstractp38 mitogen-activated protein kinases (p38 MAPKs) are a group of serine/threonine protein kinases that together with ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinases) MAPKs act to convert different extracellular signals into specific cellular responses through interacting with and phosphorylating downstream targets. In contrast to the mitogenic ERK pathway, mammalian p38 MAPK family proteins (alpha, beta, gamma, and delta), with and without JNK participation, predominantly regulate inflammatory and stress response. Recent emerging evidence suggests that the p38 stress MAPK pathway may function as a tumor suppressor through regulating Ras-dependent and -independent proliferation, transformation, invasion and cell death by isoform-specific mechanisms. A selective activation of a stress pathway to block tumorigenesis may be a novel strategy to control human malignancies. KeywordsThe p38 MAPK pathway; Ras; Tumor Suppressor; isoform-specific; Oncogenesis; Review INTRODUCTIONMAPKs (mitogen-activated protein kinases) consist of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 cascades (1, 2). Classically, MAPKs function by phosphorylating substrates containing consensus sequence Ser/Thr-Pro (3) after they are phosphorylated and activated by upstream kinases (MAPK kinases). Each of these MAPK pathways has several family members but all share the same conservative Thr-XaaTyr phosphorylation motif (where Xaa is any amino-acid) (1, 2, 4). The ERK activity is mostly frequently activated by mitogens and required for cell proliferation, differentiation and/or transformation (5, 6). JNK and p38 pathways, on the other hand, are predominantly responsive to stress and cytokine signaling and play an in important role in regulating stress response and inflammation (7,8). MAPKs can be activated almost by all type of stimuli and are consequently involved in many critical biological processes such as proliferation, differentiation, cell death and transformation through regulating downstream gene expression and/or interacting with other signaling cascades.Send correspondence to: Dr. Guan Chen, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, gchen@mcw.edu. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe p38 upstream activators include MAPK kinase 6 (MKK6) and MKK3. Downstream effectors consist of kinases such as MK2 (MAPK-activating protein kinase 2) and PRAK (p38-related/activated protein kinase) as well as transcription factors including ATF-2 (activating transcription factor-2), MEF2 (myocyte enhancement factor 2), and c-Jun (4, 9). The mammalian p38 family consists of four isoform proteins (alpha, beta, gamma, and delta), with p38alpha and p38beta 75% identical in their amino-acid sequence, and p38gamma and p38delta about 60% identical to p38alpha (4, 10). p38alpha and p38beta are susceptible to inhibition by SB drugs (SB203580 and SB202190) wh...

p38 MAPK family consists of four isoform proteins (␣, ␤, ␥, and ␦) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38␣ may regulate Ras transformation and stress response through depleting its family member p38␥ protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38␣ (MKK6-p38␣) and p38␥ (MKK6-p38␥) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38␣ phosphorylation results in either Rastransformation inhibition or a cell-death escalation that invariably couples with a decrease in p38␥ protein expression. p38␥, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38␣ phosphorylation decreases p38␥ protein expression, whereas its inhibition increases cellular p38␥ concentrations, indicating an active role of p38␣ phosphorylation in negatively regulating p38␥ protein expression. Mechanistic analyses show that p38␣ requires c-Jun activation to deplete p38␥ proteins by ubiquitin-proteasome pathways. These results suggest that p38␣ may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38␥ family protein.

The activation status of the insulin-like growth factor-1 receptor (IGF-1R) regulates the cellular response of keratinocytes to ultraviolet B (UVB) exposure, both in vitro and in vivo. Geriatric skin is deficient in IGF-1 expression resulting in an aberrant IGF-1R-dependent UVB response which contributes to the development of aging-associated squamous cell carcinoma. Furthermore, our lab and others have reported that geriatric keratinocytes repair UVB-induced DNA damage less efficiently than young adult keratinocytes. Here, we show that IGF-1R activation influences DNA damage repair in UVB-irradiated keratinocytes. Specifically, in the absence of IGF-1R activation, the rate of DNA damage repair following UVB-irradiation was significantly slowed (using immortalized human keratinocytes) or inhibited (using primary human keratinocytes). Furthermore, inhibition of IGF-1R activity in human skin, using either ex vivo explant cultures or in vivo xenograft models, suppressed DNA damage repair. Primary keratinocytes with an inactivated IGF-1R also exhibited lower steady-state levels of nucleotide excision repair mRNAs. These results suggest that deficient UVB-induced DNA repair in geriatric keratinocytes is due in part to silenced IGF-1R activation in geriatric skin and provide a mechanism for how the IGF-1 pathway plays a role in the initiation of squamous cell carcinoma in geriatric patients.

Vitamin D receptor (VDR) is a ligand-dependent transcription factor that mediates vitamin D Vitamin D receptor (VDR)4 is a ligand-dependent transcription factor and primarily functions to maintain calcium homeostasis through regulating target gene expression in response to vitamin D 3 (1). This endocrine system also regulates differentiation (2, 3), proliferation (4, 5), and cell death (6, 7) by less defined mechanisms. Moreover, vitamin D 3 has a well established cancer inhibitory activity: it prevents carcinogenesis in colon, mammary glad, and skin (8), and inhibits malignant growth in vitro and in vivo (9, 10). Whereas vitamin D 3 is generally believed to exert biological activities through its receptor VDR (the classical pathway or mechanism), it is not known whether VDR alone exhibits any of these activities in the absence of vitamin D 3 .The classical VDR pathway is believed to initiate upon vitamin D 3 binding to its receptor. Through conformation changes, VDR dimerizes with itself and/or its partners such as retinoid X receptor (RXR), which then bind to specific vitamin D responsive elements (VDREs) in a target gene promoter, leading to gene transcription alterations (1). Like other nuclear hormone receptors, VDR protein consists of several functional domains including a DNA-binding domain and a C-terminal ligandbinding domain, and functions in cooperation with co-regulators through formation of a chromatin-remodeling complex (11,12). Through signaling integrations of various target genes, vitamin D 3 /VDR activity is converted into biological effects.Recent studies suggest that vitamin D 3 and its receptor may each have distinct activities in regulating cell death. Vitamin D 3 , for example, induces cell death in some cases (7,13,14) but inhibits this event in others (15)(16)(17). Animal studies (18,19) also showed that vitamin D 3 protects against chemotherapyinduced alopecia (hair loss), a process of intrafollicular apoptosis (20). With regard to its receptor, several pieces of evidence suggest that VDR may be anti-apoptotic by itself. This was first demonstrated by induction of cell death after antisense-mediated VDR inhibition (21). Furthermore, HeLa cells stably transfected with VDR showed resistance to etoposide-induced apoptosis (6). Development of alopecia (22, 23) and increased apoptosis in periprostatic tissues (24) in VDR knock-out mice provide further genetic evidence for VDR anti-apoptotic activity. Signaling mechanisms for VDR anti-apoptotic activity as * This work was supported by National Institutes of Health NCI Grant 2R01 CA91576, a Department of Veterans Affairs Merit Review Award, and the Charlotte Geyer Foundation (to G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.