Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation

Menin uncouples Elk-1, JunD and c-Jun phosphorylation from MAP kinase activation

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Multiple members of the mitogen-activated protein kinase family are necessary for PED/PEA-15 anti-apoptotic function.

293 kidney embryonic cells feature very low levels of the anti-apoptotic protein PED. In these cells, expression of PED to levels comparable with those occurring in normal adult cells inhibits apoptosis induced by growth factor deprivation and by exposure to H(2)O(2) or anisomycin. In PED-expressing 293 cells (293(PED)), inhibition of apoptosis upon growth factor deprivation was paralleled by decreased phosphorylation of JNK1/2. In 293(PED) cells, decreased apoptosis induced by anisomycin and H(2)O(2) was also accompanied by block of JNK1/2 and p38 phosphorylations, respectively. Impaired activity of these stress kinases by PED correlated with inhibition of stress-induced Cdc-42, MKK4, and MKK6 activation. At variance with JNK1/2 and p38, PED expression increased basal and growth factor-stimulated Ras-Raf-1 co-precipitation and MAPK phosphorylation and activity. Treatment of 293(PED) cells with the MEK inhibitor PD98059 blocked ERK1/2 phosphorylations with no effect on inhibition of JNK1/2 and p38 activities. Complete rescue of JNK and p38 functions in 293(PED) cells by overexpressing JNK1 or p38, respectively, enabled only partial recovery of apoptotic response to growth factor deprivation and anisomycin. However, simultaneous rescue of JNK and p38 activities accompanied by block of ERK1/2 fully restored these responses. Thus, PED controls activity of the ERK, JNK, and p38 subfamilies of MAPKs. PED anti-apoptotic function in the 293 cells requires PED simultaneous activation of ERK1/2 and inhibition of the JNK/p38 signaling systems by PED

Crosstalk between Notch, HIF-1α and GPER in Breast Cancer EMT

The Notch signaling pathway acts in both physiological and pathological conditions, including embryonic development and tumorigenesis. In cancer progression, diverse mechanisms are involved in Notch-mediated biological responses, including angiogenesis and epithelial-mesenchymal-transition (EMT). During EMT, the activation of cellular programs facilitated by transcriptional repressors results in epithelial cells losing their differentiated features, like cell–cell adhesion and apical–basal polarity, whereas they gain motility. As it concerns cancer epithelial cells, EMT may be consequent to the evolution of genetic/epigenetic instability, or triggered by factors that can act within the tumor microenvironment. Following a description of the Notch signaling pathway and its major regulatory nodes, we focus on studies that have given insights into the functional interaction between Notch signaling and either hypoxia or estrogen in breast cancer cells, with a particular focus on EMT. Furthermore, we describe the role of hypoxia signaling in breast cancer cells and discuss recent evidence regarding a functional interaction between HIF-1α and GPER in both breast cancer cells and cancer-associated fibroblasts (CAFs). On the basis of these studies, we propose that a functional network between HIF-1α, GPER and Notch may integrate tumor microenvironmental cues to induce robust EMT in cancer cells. Further investigations are required in order to better understand how hypoxia and estrogen signaling may converge on Notch-mediated EMT within the context of the stroma and tumor cells interaction. However, the data discussed here may anticipate the potential benefits of further pharmacological strategies targeting breast cancer progression

Detection and toxigenic evaluation of endophytic strains of Aspergillus flavus from Italian rice seeds

Ten strains of A. flavus, all characterized by a very slow rate of hyphal production from the plated seeds (from two to four weeks of incubation), were respectively isolated from ten cultivars. In particular, seven strains were detected from shattered seeds (belonging to cvs. Ares, Belgioioso, Delfino, Elvo, Idra, Maratelli, Venere) and three strains from sterilized but not broken seeds (cvs. Dedalo, Gemini, Perseo). As different seeds of the same cultivar were always endophytically colonized by the species, results were expressed as per cent number of colonized seeds. The highest percentages of A. flavus were detected in seeds of cvs. Belgioioso (50%), Delfino (39%), Maratelli (30%) and Idra (21%); the lowest in seeds of cvs. Perseo (8%), Dedalo (6%), Ares (6%), Gemini (4%), Venere (3%), Elvo (2%). When transferred onto PDA medium, all the strains started to produce a large quantity of brown to black, globose to subglobose, about 500 µm in diameter sclerotia. Supported by an extremely positive correlation between the production of both sclerotia and mycotoxins, individuated in some atypical A. flavus studied by Saito et al. (1986), the toxigenic activity of all rice strains was analyzed

The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17beta-estradiol and hydroxytamoxifen in endometrial cancer cells

The growth of both normal and transformed epithelial cells of the female reproductive system is stimulated by estrogens, mainly through the activation of estrogen receptor alpha (ERalpha), which is a ligand-regulated transcription factor. The selective ER modulator tamoxifen (TAM) has been widely used as an ER antagonist in breast tumor; however, long-term treatment is associated with an increased risk of endometrial cancer. To provide new insights into the potential mechanisms involved in the agonistic activity exerted by TAM in the uterus, we evaluated the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, to transactivate wild-type ERalpha and its splice variant expressed in Ishikawa and HEC1A endometrial tumor cells, respectively. OHT was able to antagonize only the activation of ERalpha by 17beta-estradiol (E2) in Ishikawa cells, whereas it up-regulated c-fos expression in a rapid manner similar to E2 and independently of ERalpha in both cell lines. This stimulation occurred through the G protein-coupled receptor named GPR30 and required Src-related and epidermal growth factor receptor tyrosine kinase activities, along with the activation of both ERK1/2 and phosphatidylinositol 3-kinase/AKT pathways. Most importantly, OHT, like E2, stimulated the proliferation of Ishikawa as well as HEC1A cells. Transfecting a GPR30 antisense expression vector in both endometrial cancer cell lines, OHT was no longer able to induce growth effects, whereas the proliferative response to E2 was completely abrogated only in HEC1A cells. Furthermore, in the presence of the inhibitors of MAPK and phosphatidylinositol 3-kinase pathways, PD 98059 and wortmannin, respectively, E2 and OHT did not elicit growth stimulation. Our data demonstrate a new mode of action of E2 and OHT in endometrial cancer cells, contributing to a better understanding of the molecular mechanisms involved in their uterine agonistic activity

The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells

A growing body of evidence concerning estrogen effects cannot be explained by the classic model of hormone action, which involves the binding to estrogen receptors (ERs) alpha and ERbeta and the interaction of the steroid-receptor complex with specific DNA sequences associated with target genes. Using c-fos proto-oncogene expression as an early molecular sensor of estrogen action in ERalpha-positive MCF7 and ER-negative SKBR3 breast cancer cells, we have discovered that 17beta-estradiol (E2), and the two major phytoestrogens, genistein and quercetin, stimulate c-fos expression through ERalpha as well as through an ER-independent manner via the G protein-coupled receptor homologue GPR30. The c-fos response is repressed in GPR30-expressing SKBR3 cells transfected with an antisense oligonucleotide against GPR30 and reconstituted in GPR30-deficient MDA-MB 231 and BT-20 breast cancer cells transfected with a GPR30 expression vector. GPR30-dependent activation of ERK1/2 by E2 and phytoestrogens occurs via a Gbetagamma-associated pertussis toxin-sensitive pathway that requires both Src-related and EGF receptor tyrosine kinase activities. The ability of E2 and phytoestrogens to regulate the expression of growth-related genes such as c-fos even in the absence of ER has interesting implications for understanding breast cancer progression

17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17beta-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERalpha that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC