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YAP signaling has emerged as an important signaling pathway involved in several normal and pathological processes. While main upstream effectors regulating its activity have been extensively studied, the interplay with other cellular processes has been far less analyzed. Here, we identified the LAMTOR complex as a new important regulator of YAP signaling. We uncovered that p18/LAMTOR1 is required for the recycling of Src on late endosomes to the cell periphery, and consequently to activate a signaling cascade that eventually controls YAP nuclear shuttling. Moreover, p18/LAMTOR1 positives late endosomes distribution is controlled by β1 integrins, extracellular matrix stiffness and cell contractility. This likely relies on the targeting of microtubules to β1 positive focal adhesion via ILK. Altogether our findings identify the late endosomal recycling pathway as a major regulator of YAP.

International audience; Although Merlin function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt or Hippo pathways is well documented,in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora A kinase during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in Hela cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates Ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with Neurofibromatosis type 2 that display an aberant phosphorylation profile along with defective α-tubulin binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and Ezrin suggest a potential role for Merlin in cell cycle progression.

BACKGROUND: Clinical overlap between neurofibromatosis type 2 (NF2), schwannomatosis, and meningiomatosis can make clinical diagnosis difficult. Hence, molecular investigation of germline and tumor tissues may improve the diagnosis. METHODS: We present the targeted next-generation sequencing (NGS) of NF2, SMARCB1, LZTR1, SMARCE1, and SUFU tumor suppressor genes, using an amplicon-based approach. We analyzed blood DNA from a cohort of 196 patients, including patients with NF2 (N = 79), schwannomatosis (N = 40), meningiomatosis (N = 12), and no clearly established diagnosis (N = 65). Matched tumor DNA was analyzed when available. Forty-seven NF2-/SMARCB1-negative schwannomatosis patients and 27 NF2-negative meningiomatosis patients were also evaluated. RESULTS: A NF2 variant was found in 41/79 (52%) NF2 patients. SMARCB1 or LZTR1 variants were identified in 5/40 (12.5%) and 13/40 (∼32%) patients in the schwannomatosis cohort. Potentially pathogenic variants were found in 12/65 (18.5%) patients with no clearly established diagnosis. A LZTR1 variant was identified in 16/47 (34%) NF2/SMARCB1-negative schwannomatosis patients. A SMARCE1 variant was found in 3/39 (∼8%) meningiomatosis patients. No SUFU variant was found in the cohort. NGS was an effective and sensitive method to detect mutant alleles in blood or tumor DNA of mosaic NF2 patients. Interestingly, we identified a 4-hit mechanism resulting in the complete NF2 loss-of-function combined with SMARCB1 and LZTR1 haploinsufficiency in two-thirds of tumors from NF2 patients. CONCLUSIONS: Simultaneous investigation of NF2, SMARCB1, LZTR1, and SMARCE1 is a key element in the differential diagnosis of NF2, schwannomatosis, and meningiomatosis. The targeted NGS strategy is suitable for the identification of NF2 mosaicism in blood and for the investigation of tumors from these patients.

Biallelic inactivation of the NF2 tumor suppressor gene leads to the development of intracranial tumors such as schwannomas, meningiomas, and ependymomas. These lesions can be sporadic or develop in the context of an inherited familial disease called neurofibromatosis type 2. Patients who are born heterozygous for NF2 are predisposed to the development of multiple tumors upon subsequent loss of the second allele.1 To this day, surgery and radiotherapy remain the most frequent options for treatment. The use of chemotherapy has long been hindered by the lack of clearly identified therapeutic targets. Since the discovery of the NF2 gene in 1993,2 a major effort has been made to understand how merlin, the NF2 gene product, regulates cell proliferation and tumor growth. Several proteins playing key roles in these processes have emerged as candidate therapeutic targets. The inactivation of NF2 was shown to trigger the activation of Ras and Rac and the stimulation of downstream mitogenic p42/44 MAPK and PAK1/2 pathways.3,4 It has also been demonstrated that loss of merlin expression induces the activity and surface expression of various receptor tyrosine kinases (RTKs). In the latter case, it is due to an increase of their transport to the plasma membrane,5–7 leading to stimulation of promitogenic signaling pathways. Recently, a new function for merlin in the nucleus was discovered.8 In this compartment, the binding of merlin to D-Caf1 abolishes its ubiquitin ligase activity and leads to the inhibition of proliferation. Finally, the regulation of the Hippo signaling pathway by merlin has emerged in recent years as a major mechanism controlling cell proliferation and survival in various organisms and tissues.9,10 Indeed, in several models, loss of merlin expression leads to nuclear accumulation of Yes-associated protein (YAP), which is the major effector of the Hippo pathway.11,12 There, it stimulates transcription of a wide set of promitogenic and anti-apoptotic target genes.13 Hence, the growth advantage conferred by the loss of NF2 appears to be the consequence of an accumulation of distinct signaling dysfunctions. The relevance of these mechanisms of growth control by merlin has also been tested on tumor cell cultures derived from human biopsies. Receptors such as IGF1R, Axl, or PDGFRs and signaling pathways such as MAPK, Rac/PAK, JNK, or β-catenin were shown to be activated in response to merlin loss of expression.14–16 However, the tumor cell cultures always represent a simplified model of their tumor of origin. In this context, evaluation of the expression and activity of signaling pathways in surgical biopsies represents a necessary complement to the previous approaches. Immunohistological evaluation of the expression and activity of signaling proteins in tumor sections has provided important information on the biology of NF2-related tumors.17–19 Nevertheless, new proteomic approaches such as reverse phase protein arrays (RPPA) allow more precise means for quantifying signaling protein expression and activity on large sets of biological samples.20 Therefore, combining different types of proteomic analysis for the study of tumors constitutes a powerful approach for identifying the signaling events that promote their growth. Using a set of more than 40 human schwannomas, we have analyzed the status of several major signaling pathways that were previously shown to be regulated by merlin. First, using receptor tyrosine kinase (RTK) arrays and Western blotting, we have identified which RTKs are the most frequently activated in schwannomas and could therefore represent a potential therapeutic target. Then, RPPA was used on our tumor set to measure the expression and activity of 35 proteins comprising the major mitogenic signaling pathways. Our results showed that expression levels of YAP and several of its target genes, notably several receptors identified through RTK arrays, are linked to proliferation. However, immunohistochemical analysis revealed a strong variability of protein expression from tumor to tumor, suggesting that the response to targeted chemotherapy would likely be highly variable. Altogether, our work identifies a signaling network associated with YAP protein and linked to schwannoma cell proliferation that could represent a set of valuable therapeutic targets. Statement of translational relevance Patients affected by the familial syndrome Neurofibromatosis type 2 develop multiple tumors when the NF2 suppressor gene is inactivated. Schwannomas are the most frequent ones and the number of chemotherapeutic options is yet very limited. Hence, the identification of potential therapeutic targets is a major objective of the research effort on this tumor type. In this study, we have combined several proteomic approaches to investigate the expression and activity of mitogenic signaling pathways regulated by NF2. Our work shows that proliferation of tumor cells is correlated to the activation of a signaling network linked to the Hippo effector Yap and several of its target genes. Hence, our study provides evidences that Yap as well as its target genes PDGFRs, Her3, Her2 and Axl represent potentially new therapeutic targets for the treatment of schwannomas.

International audience; Cell adhesion to the extracellular matrix or to surrounding cells plays a key role in cell proliferation and differentiation, and is critical for proper tissue homeostasis. An important pathway in adhesion-dependent cell proliferation is the Hippo signaling cascade, which is coregulated by the transcription factors Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ). However, how cells integrate extracellular information at the molecular level to regulate YAP1's nuclear localization is still puzzling. Herein, we investigated the role of β1 integrins in regulating this process. We found that β1 integrin–dependent cell adhesion is critical for supporting cell proliferation in mesenchymal cells both in vivo and in vitro. β1 integrin– dependent cell adhesion relied on the relocation of YAP1 to the nucleus after the downregulation of its phosphorylated state mediated by large tumor suppressor gene 1 and 2 (LATS1/2). We also found that this phenotype relies on β1 integrin–dependent local activation of the small GTPase Rac1 at the plasma membrane to control the activity of P21 (RAC1)-activated kinase (PAK) of group 1. We further report that the regulatory protein merlin (neurofibromin 2, NF2) interacts with both YAP1 and LATS1/2 via its C-terminal moiety and FERM domain, respectively. PAK-mediated merlin phosphorylation on Ser-518 reduced merlin's interactions with both LATS1/2 and YAP1, resulting in YAP1 dephosphorylation and nuclear shuttling. Our results highlight Rac1/PAK1 as major players in YAP1 regulation triggered by cell adhesion.

The NF2 gene product, merlin/schwannomin, is a cytoskeleton organizer with unique growth-inhibiting activity in specific cell types. A narrow spectrum of tumors is associated with NF2 deficiency, mainly schwannomas and meningiomas, suggesting cell-specific mechanisms of growth control. We have investigated merlin function in mouse Schwann cells (SCs). We found that merlin regulates contact inhibition of proliferation by limiting the delivery of several growth factor receptors at the plasma membrane of primary SCs. Notably, upon cell-to-cell contact, merlin downregulates the membrane levels of ErbB2 and ErbB3, thus inhibiting the activity of the downstream mitogenic signaling pathways protein kinase B and mitogen-activated protein kinase. Consequently, loss of merlin activity is associated with elevated levels of ErbB receptors in primary SCs. We also observed accumulation of growth factor receptors such as ErbB2 and 3, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor in peripheral nerves of Nf2-mutant mice and in human NF2 schwannomas, suggesting that this mechanism could play an important role in tumorigenesis.

The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a membrane/cytoskeleton-associated protein that mediates contact-dependent inhibition of proliferation. Here we show that upon cell–cell contact Merlin coordinates the processes of adherens junction stabilization and negative regulation of epidermal growth factor receptor (EGFR) signaling by restraining the EGFR into a membrane compartment from which it can neither signal nor be internalized. In confluent Nf2−/− cells, EGFR activation persists, driving continued proliferation that is halted by specific EGFR inhibitors. These studies define a new mechanism of tumor suppression, provide mechanistic insight into the poorly understood phenomenon of contact-dependent inhibition of proliferation, and suggest a therapeutic strategy for NF2-mutant tumors.

Mutation of theNeurofibromatosis 2(NF2) tumor suppressor gene leads to cancer development in humans and mice. Recent studies suggest thatNf2loss also contributes to tumor metastasis. TheNf2-encoded protein, merlin, is related to the ERM (ezrin,radixin, andmoesin) family of membrane:cytoskeleton-associated proteins. However, the cellular mechanism whereby merlin controls cell proliferation from this location is not known. Here we show that the major cellular consequence ofNf2deficiency in primary cells is an inability to undergo contact-dependent growth arrest and to form stable cadherin-containing cell:cell junctions. Merlin colocalizes and interacts with adherens junction (AJ) components in confluent wild-type cells, suggesting that the lack of AJs and contact-dependent growth arrest inNf2−/−cells directly results from the absence of merlin at sites of cell:cell contact. Our studies indicate that merlin functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell:cell contact.

Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.

Theileria parasitises bovine leukocytes and transforms them into proliferating, metastatic tumours, where the infection resembles a leukaemia-like disease. We have studied the signal transduction pathways leading to activation of the transcription factor AP-1 in different transformed leukocytes. Parasite infection leads to an up-regulation of all members of the Jun/Fos family of proteins and surprisingly, this occurs in the absence of any detectable ERK, or p38 MAP kinase activity. In the parasitised B-sarcoma TBL3, AP-1 induction occurs in the absence of any JNK activity. In contrast, in infected macrophage and B-cell lines, AP-1 transcriptional activity is strictly associated with the parasite-induced constitutive activation of JNK and subsequent c-Jun N-terminal phosphorylation. Thus, constant AP-1 transcriptional activity involves both an upregulation in the levels of Jun and Fos proteins and constitutive JNK activation.

The Ras proteins play a central role in regulating cell growth and their mutation can lead to abnormal proliferation. To analyse the potential link betwen AP1 activity, encoded by members of the jun and fos gene families, and Ras-mediated cellular transformation, we have studied several NIH3T3 clones which overexpress the Ha-Ras or Ki-Ras oncogenes. These transformed fibroblasts accumulated higher levels of cJun, JunB, Fra1 and Fra2 proteins relative to their normal counterparts. They also displayed increased AP1 DNA binding activity which was predominantly composed of cJun and Fra1 containing dimers. Following serum stimulation of Ras clones, the elevated levels of cJun and Fral remained steady, while the induction of JunB and Fra2 was partially attenuated. Moreover, deregulated Ras signaling resulted in a complete loss of the serum inducibility of cFos and FosB. Ectopic co-expression of cJun and Fra1 in NIH3T3 fibroblasts led to a transformed phenotype, attenuation of cFos serum inducibility, increased AP1 activity and Cyclin D1 accumulation, all characteristics of oncogenic Ras expressing cells. These results demonstrate that cJun and Fra1 are crucial mediators of the Ras-transformation process.

We have analysed the different Jun and Fos proteins as NIH3T3 fibroblasts pass from exponential growth to quiescence and during the first 24 h after their re-entry into the cell cycle following serum stimulation. We show that these proteins can be divided into 3 subgroups based on their pattern of expression. The first contains c-Jun, Jun-D and Fra-2 which are expressed at high level in cycling cells and are only mildly induced by serum. The second contains Jun-B, c-Fos, Fos-B and deltaFos-B whose levels are low in cycling cells but increase strongly and rapidly after stimulation by serum. The third group contains only Fra-1, which is absent from cycling cells and behaves as a delayed early response protein after serum stimulation. AP-1 binding activity is low both in cycling and quiescent fibroblasts but increases after stimulation by serum with kinetics matching the induction of the various Jun and Fos proteins. Antibody supershift analyses demonstrate that the composition of AP-1 binding activity reflects the relative abundance of each Jun and Fos protein. Furthermore, the state of post-translational modification varies continuously for all of the AP-1 proteins as growth conditions change. These data indicate that AP-1 activity during the G0-G1 transition is finely regulated and complex, involving changes both in protein expression and in posttranslational modification.

The French national agency for the assessment and quality of social and medical-social institutes and services (ANESM) recently published recommendations aiming to promote the quality of life of elderly people in nursing homes. These recommendations take into account the specific characteristics of the quality of life of this sector of the population.

Merlin is the product of the Nf2 tumor-suppressor gene, and inactivation of Nf2 leads to the development of neural tumors such as schwannomas and meningiomas in humans and mice. Merlin is a member of the ERM (ezrin, radixin and moesin) family of proteins that function as organizers of the actin cytoskeleton. Merlin structure is thought to be similar to that of the ERM proteins, and is held in a closed clamp conformation via intramolecular interactions of its N-terminal FERM (four-point-one, ERM) domain with an α-helical C-terminal domain. Like ERMs, merlin can remodel actin-rich cortical structures, yet merlin uniquely inhibits the proliferation of many different cell types. Here, we report that the F2 subdomain of the FERM domain and a domain close to the C-terminus that is defined by residues 532-579 are essential for merlin-mediated inhibition of primary Schwann cell proliferation. Furthermore, we demonstrate that the F1 subdomain of the merlin FERM domain is required for actin colocalization, proper regulation of merlin C-terminal phosphorylation and for remodeling the cytoskeleton, yet is not required for the inhibition of Schwann cell proliferation. Thus, tumor suppression by merlin is independent of its role as an organizer of the actin cytoskeleton in Schwann cells.

The lack of neurofibromatosis 2 tumor suppressor protein merlin leads to the formation of nervous system tumors, specifically schwannomas and meningiomas. Merlin is considered to act as a tumor suppressor at the cell membrane, where it links transmembrane receptors to the actin cytoskeleton. Several tumor suppressors interact with another component of the cytoskeleton, the microtubules, in a regulated manner and control their dynamics. In this work, we identify merlin as a novel microtubule-organizing protein. We identify two tubulin-binding sites in merlin, one residing at the N-terminal FERM-domain and another at the C-terminal domain. Merlin's intramolecular association and phosphorylation of serine 518 regulate the interaction between merlin and tubulin. Analysis of cultured glioma cells indicates colocalization between merlin and microtubules especially during cell division. In primary mouse Schwann cells only minor colocalization at the cell periphery of interphase cells is seen. However, these cells drastically change their microtubule organization upon loss of merlin indicating a functional association of the proteins. Both in vitro assays and in vivo studies in Schwann cells indicate that merlin promotes tubulin polymerization. The results show that merlin plays a key role in the regulation of the Schwann cell microtubule cytoskeleton and suggest a mechanism by which loss of merlin leads to cytoskeletal defects observed in human schwannomas.

The transcription factor AP‐1, composed of Jun and Fos proteins, is a major target of mitogen‐activated signal transduction pathways. However, little is known about AP‐1 function in normal cycling cells. Here we report that the quantity and the phosphorylation state of the c‐Jun and JunB proteins vary at the M–G 1 transition. Phosphorylation of JunB by the p34 cdc2 –cyclin B kinase is associated with lower JunB protein levels in mitotic and early G 1 cells. In contrast, c‐Jun levels remain constant while the protein undergoes N‐terminal phosphorylation, increasing its transactivation potential. Since JunB represses and c‐Jun activates the cyclin D1 promoter, these modifications of AP‐1 activity during the M–G 1 transition could provide an impetus for G 1 progression by a temporal increase in cyclin D1 transcription. These findings constitute a novel example of a reciprocal connection between transcription factors and the cell cycle machinery.

Barsh, G.S. (2006). Regulation of pigment type switching by agouti, melanocortin signaling, attractin and mahoganoid. In The Pigmentary System, 2nd edn. J.J. Nordlund, R.E. Boissy, V.J. Hearing, R.A. King, W.S. Oetting, and J.P. Ortonne, eds (Massachusetts: Blackwell Publishing), pp. 395–409. Candille, S.I., Kaelin, C.B., Cattanach, B.M., Yu, B., Thompson, D.A., Nix, M.A., Kerns, J.A., Schmutz, S.M., Millhauser, G.L., and Barsh, G.S. (2007). A b-defensin mutation causes black coat color in domestic dogs. Science 318, 1418–1423. Chakrabarti, O., and Hegde, R.S. (2009). Functional depletion of mahogunin by cytosolically exposed prion protein contributes to neurodegeneration. Cell 137, 1136–1147. Hida, T., Wakamatsu, K., Sviderskaya, E.V. et al. (2009). Agouti protein, mahogunin, and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes: a cAMP-independent pathway. Pigment Cell Melanoma Res. 22, 623–634. Jiao, J., Kim, H.Y., Liu, R.R., Hogan, C.A., Sun, K., Tam, L.M., and Gunn, T.M. (2009). Transgenic analysis of the physiological functions of Mahogunin Ring Finger-1 isoforms. Genesis 47, 524–534.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is highly conserved within vertebrate species. Its pattern of expression in vivo seems to be tightly regulated both developmentally and in a tissue-specific manner, but shows differences with species. To identify transcriptional regulatory elements in the CFTR promoter region, we have used a combined approach based both on the analysis of the chromatin structure in vivo in rat tissues and on evolutionary clues (i.e. phylogenetic footprinting). In CFTR-expressing tissues, 15 DNase I-hypersensitive sites were identified within a 36 kb region encompassing exon 1. Eleven of them are clustered in a 3.5 kb region that exhibits eleven phylogenetic footprints observed when comparing sequences from eight mammalian species representing four orders (Primates, Artiodactylia, Lagomorpha and Rodentia). Comparison of the two sets of data allows the identification of two types of regulatory elements. Some are conserved between species, such as a non-consensus cAMP response element (CRE) and a PMA-responsive element (TRE) located respectively at positions -0.1 and -1.3 kb relative to ATG. Some are species-specific elements such as a 300 bp purine·pyrimidine (Pu·Py) stretch that is present only in rodents. Analysis of protein/DNA interactions in vitro with rat tissue protein extracts on the conserved elements revealed that the TRE site binds a specific heterodimeric complex composed of Fra-2, Jun D and a protein immunologically related to Jun/CRE-binding protein in the duodenum, whereas the CRE-like site binds ATF-1 ubiquitously. Functional analysis in Caco-2 cells showed that the CRE-like site supports a high basal transcriptional activity but is not able by itself to induce a response to cAMP, whereas the TRE site acts as a weak transactivator stimulated by PMA. Lastly, we found that the rodent-specific Pu·Py stretch confers nuclease S1 hypersensitivity under conditions of acidic pH and supercoiling. This indicates a non-B DNA conformation and thus reinforces the biological significance of non-random Pu·Py strand asymmetry in the regulation of transcription. Thus the tight transcriptional regulation of CFTR expression involves the combination of multiple regulatory elements that act in the chromatin environment in vivo. Some of them are conserved throughout evolution, such as the CRE-like element, which is clearly involved in the basal level of transcription; others are species-specific.

In mammalian melanocytes, melanin synthesis is controlled by tyrosinase, the critical enzyme in the melanogenic pathway. We and others showed that the stimulation of melanogenesis by cAMP is due to an increased tyrosinase expression at protein and mRNA levels. However, the molecular events connecting the rise of intracellular cAMP and the increase in tyrosinase activity remain to be elucidated. In this study, using B16 melanoma cells, we showed that cAMP-elevating agents stimulated mitogen-activated protein (MAP) kinase, p44mapk. This effect was mediated by the activation of MAP kinase kinase. cAMP-elevating agents induced a translocation of p44mapk to the nucleus and an activation of the transcription factor AP-1. cAMP-induced AP-1 contained FOS-related antigen-2 in association with JunD, while after phorbol ester stimulation AP-1 complexes consist mainly of JunD/c-Fos heterodimers. In an attempt to connect these molecular events to the control of tyrosinase expression that appears to be the pivotal point of melanogenesis regulation, we hypothesized that following its activation by cAMP, p44mapk activates AP-1. Then AP-1 could stimulate tyrosinase expression through the interaction with specific DNA sequences present in the mouse tyrosinase promoter.

Sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis in its absence. We have investigated the pattern of expression of the Jun and Fos family of transcription factors in dying sympathetic neurons using antibodies specific for each family member. When sympathetic neurons are deprived of NGF, the level of c-Jun protein significantly increases, whereas the levels of the other members of the Jun and Fos family remain relatively constant. c-Jun also becomes more phosphorylated, probably on its amino terminal transactivation domain. When microinjected into sympathetiic neurons, an expression vector for a c-Jun dominant negative mutant protects them against NGF withdrawal-induced death, indicating that AP-1 activity is essential for neuronal cell death. Furthermore, overexpression of the full-length c-Jun protein is, in itself, sufficient to induce apoptosis in sympathetic neurons.

We have measured the level of junB mRNA in the B hybridoma cell line 7TD1, under interleukin-6 (IL-6) stimulation. IL-6 increases junB mRNA in a biphasic fashion. The first early-induced peak was transient and likely corresponds to the well documented typical junB mRNA, stimulated in response to numerous growth factors, including IL-6. At variance, the second peak which has never been reported previously, lasted several hours. As a consequence of its effect on junB mRNA, IL-6 stimulated, in a biphasic fashion, the nuclear accumulation of the JunB protein. In this study, we demonstrated that IL-6 regulation occurred exclusively at the transcriptional level and that the bimodal increase of junB mRNA and JunB protein can be accounted for by a biphasic stimulation of junB transcription. Furthermore, our data point to two major differences between the mechanism of control of the early and the late IL-6-induced junB transcription waves. First, cycloheximide strongly potentiated the transcription of the second wave, whereas it failed to affect the early-induced burst. Second, tyrphostin, a tyrosine kinase inhibitor, impaired the expression of the first but not the second junB mRNA peak. Conversely, genistein, another tyrosine kinase inhibitor, totally abolished the expression of the second peak of junB mRNA whereas it did not affect the expression of the first peak. Altogether these data indicate that, in 7TD1 cells, IL-6 controls junB transcription in a biphasic fashion by means of two separate transduction pathways.

As NIH 3T3 fibroblasts become quiescent, the level of c-Jun protein decreases while JunD accumulates. When resting cells are stimulated with fresh serum, nuclear-localized JunD is rapidly degraded, followed by resynthesis of both c-Jun and JunD later in G1. Overexpression of JunD results in slower growth and an increase in the percentage of cells in G0/G1 while c-Jun overexpression produces larger S/G2 and M phase populations. In addition, JunD partially suppresses transformation by an activated ras gene whereas c-Jun cooperates with ras to transform cells. These data indicate that two closely related transcription factors can function in an opposing manner.

Lovastatin-induced inhibition of renal epithelial tubular cell proliferation involves a p21 ras activated, AP-1-dependent pathway. Proliferation of tubular epithelial cells underlies the development of cystic lesions and the subsequent impairment of renal function after renal mass reduction. The effect of HMG CoA reductase inhibitors (HRI) on cell proliferation was investigated in rat renal proximal tubular epithelial cells in primary culture. Treatment of renal tubular epithelial cells with three different HRI reduced fetal calf serum (FCS)-induced [ 3 H]-thymidine incorporation (IC 50 values were 0.7 µ M, 1.7 µ M, and 1.6 µ M for simvastatin, lovastatin, and compactin, respectively), and lovastatin blocked BrdUrd incorporation, as assessed by immunocytochemical studies. The proliferative effect of epidermal growth factor (EGF) was similarly abolished by lovastatin. The effect of lovastatin (1 µ M) was prevented by 100 µ M mevalonate, 5 µ M farnesyl-pyrophosphate and 5 µ M geranylgeranyl-pyrophosphate (in percent of control value, 31% vs. 102%, 60%, and 82%, respectively) while cholesterol and other products of the mevalonate pathway were inactive. Immunoblot analysis showed that lovastatin decreased membrane-bound p21 ras and inhibited FCS-induced c-fos and c-jun protein expression. Furthermore, electrophoretic mobility shift assay demonstrated the functional impairement of AP-1 DNA binding activity in lovastatin-treated cells. In conclusion, these results demonstrate that HRI are antiproliferative in epithelial tubule cells and that this effect is exerted, at least in part, via inhibition of the p21 ras -activated and AP-1 dependent mitogenic cascade.

Interleukin-6 (IL-6) is a proinflammatory cytokine which also acts as a growth factor for some murine hybridomas (7TD1) or human myelomas (U266). We demonstrate that elevation of cAMP cellular content inhibits IL-6-stimulated cell growth, by blocking cells mainly in G1 phase. This inhibition is associated with increased expression of the Fos family protein Fra-2. Treatment of cells with 8Br-cAMP results in increased DNA-binding activity of two distinct AP-1 complexes; JunD/Fra-2 and JunB/Fra-2, and also in elevated AP-1 transactivation. When 8Br-cAMP is withdrawn from the medium, cells enter S phase and Fra-2 protein levels and AP-1 DNA-binding activity decrease to their basal value indicating that a temporally correlation exists between the 8Br-cAMP-mediated induction of JunD/Fra-2 AP-1 complex and the 7TD1 and U266 cell growth inhibition.

Cerebellar granule neurons die by apoptosis when deprived of survival signals. This death can be blocked by inhibitors of transcription or protein synthesis, suggesting that new gene expression is required. Here we show thatc-junmRNA and protein levels increase rapidly after survival signal withdrawal and that transfection of the neurons with an expression vector for a c-Jun dominant negative mutant protects them against apoptosis. Phosphorylation of serines 63 and 73 in the c-Jun transactivation domain is known to increase c-Jun activity. By using an antibody specific for c-Jun phosphorylated on serine 63, we show that this site is phosphorylated soon after survival signal withdrawal. To determine whether c-Jun phosphorylation is necessary for apoptosis, we have expressed c-Jun phosphorylation site mutants in granule neurons. c-Junasp, a constitutively active c-Jun mutant in which the known and potential serine and threonine phosphoacceptor sites in the transactivation domain have been mutated to aspartic acid, induces apoptosis under all conditions tested. In contrast, c-Junala, which cannot be phosphorylated because the same sites have been mutated to alanine, blocks apoptosis caused by survival signal withdrawal. Finally, we show that cerebellar granule neurons contain high levels of Jun kinase activity and low levels of p38 kinase activity, neither of which increases after survival signal withdrawal. Mitogen-activated protein kinase activity decreases under the same conditions. These results suggest that c-Jun levels and c-Jun phosphorylation may be regulated by novel mechanisms in cerebellar granule neurons.

Neurofibromatosis type 2 (NF2) is an autosomal disorder caused by inactivation of NF2 gene or loss of the NF2 product, Merlin. This in turn results in formation of multiple benign (noninvasive) nerve tumors such as schwannomas, meningiomas and ependymomas. Additionally reduced expression of Merlin is observed in invasive breast cancers however the role of Merlin in these invasive tumors is poorly investigated. Merlin is the only tumor suppressor protein in Ezrin/Radixin/Moesin (ERM) family proteins. Previously we and others have shown that C-terminus of Merlin is important for its growth suppressive function. In this regard, I set out to investigate whether there were undiscovered interacting partners and novel phosphorylation sites on the C-terminus of Merlin that could account for tumor suppressor function of Merlin. Using immunoprecipitation coupled to mass spectrometry we have identified new interactors as well as novel phosphorylation on this C-terminus domain of Merlin. We analyzed importance of new interactor, AmotL1, as well as novel phosphorylation site on T581 in the tumor suppressor function of Merlin. AmotL1 belongs to AMOT family proteins which are known to involve in the regulation of cell migration. In this regard, we have shown that AmotL1 is novel interacting partner of Merlin. We have investigated the importance of Merlin and AmotL1 interactions in cell migration and our data strongly suggest that Merlin might inhibit AmotL1 mediated cell migration in breast cancer cells by regulating its expression and localization. Finally, we have also found several new interactors of Merlin and that could explain how Merlin might acts as scaffolding protein at the plasma membrane by interacting with Hippo core components such as AmotL1, Kibra, Lats and YAP to regulate cell proliferation and migration. In the second part, we have identified a novel phosphorylation site at T581 which is specific to Merlin isoform 1 and demonstrated that phosphorylation of Merlin on T581 is important for the timely mitotic progression. Further in this study, we have shown that Merlin is a potential substrate for major oncogene Aurora kinase A in mitosis as well as in interphasic breast cancer cell lines. Finally we have provided initial clues how Aurora A regulates Hippo signaling and DCAF1 function by phosphorylating Merlin. In the summary, this thesis highlights two important functions of Merlin: firstly how Merlin regulates the cell migration/invasion in non-nerve tumors such as breast cancers and secondly how Merlin is regulated in mitosis and interphasic breast cancer cells by acting as a substrate to Aurora Kinase A which is over expressed in several cancers such as breast, colon and HCC. All together our study indicates the potential role for Merlin in invasive tumors such as breast cancers., La neurofibromatose de type 2 (NF2) est une maladie autosomique causée soit par l'inactivation du gène NF2, soit par la perte de la protéine issue due ce gène, Merline. Cela entraîne à son tour la formation de plusieurs tumeurs nerveuse bénignes (non invasives) comme les schwannomes, méningiomes et les épendymomes. De plus, une diminution de l'expression de Merline est observée dans les cancers du sein invasifs, toutefois le rôle de Merline dans ces tumeurs invasives est peu étudié. Merline est la seule protéine ayant un rôle de suppresseur de tumeur dans la famille des ERM (Ezrin / Radixin / Moesin). Nous, ainsi que d'autres groupes, avons montré que la partie C-terminale de Merline est importante pour sa fonction inhibitrice de la croissance cellulaire. Par conséquent, j'ai cherché à mettre en évidence de nouveaux partenaires d'interaction non décrits à ce jour, ainsi que de nouveaux sites de phosphorylation sur l'extrémité C-terminale de Merline qui pourrait expliquer la fonction de suppresseur de tumeur de Merlin. L'utilisation d’expériences d'immunoprécipitation couplées à la spectrométrie de masse nous a permis d’identifier de nouveaux interacteurs ainsi que de nouveaux sites de phosphorylation sur ce domaine C-terminal de Merline. Nous avons analysé l'importance d'un nouvel interacteur, AmotL1, ainsi que d'un nouveau site de phosphorylation sur la threonine 581 (T581), dans la fonction suppresseur de tumeur de Merline. La protéine AmotL1 appartient à la famille des motines, qui sont connues pour être impliquées dans la régulation de la migration cellulaire. A cet égard, nous avons montré qu’AmotL1 est un nouveau partenaire d'interaction de Merline. Nous avons étudié l'importance de cette interaction entre Merline et AmotL1 dans la migration cellulaire et nos données suggèrent fortement que Merlin pourrait inhiber la migration cellulaire médiée par AmotL1 dans les cellules du cancer du sein, via notamment la régulation de son expression et de sa localisation. Enfin, nous avons également identifié plusieurs nouveaux interacteurs de Merline, qui pourraient expliquer comment Merlin pourrait agir comme une protéine d'échafaudage à la membrane plasmique, en interagissant avec des composants essentiels de la voie Hippo, comme AmotL1, Kibra, Lats et YAP, pour réguler la prolifération et la migration cellulaire. Dans la deuxième partie, nous avons identifié un nouveau site de phosphorylation spécifique à l'isoforme 1 de Merline, la T581, et nous avons démontré que la phosphorylation de cette threonine est importante pour la progression en mitose au moment approprié. De plus, dans cette étude, nous avons montré que Merlin est un substrat potentiel de la kinase Aurora A, un oncogène majeur, au cours de la mitose et de l'interphase, dans des lignées cellulaires de cancer du sein. Enfin, nous avons fourni des données préliminaires sur la façon dont Aurora A régule la signalisation Hippo et la fonction de DCAF1 en phosphorylant Merline. En résumé, cette thèse met en évidence deux fonctions importantes de Merline : premièrement comment Merline régule la migration/invasion cellulaire dans des tumeurs non-nerveuses telles que les cancers du sein et deuxièmement, comment Merline est régulé au cours de la mitose et de l'interphase dans des lignées de cancer du sein, en agissant comme un substrat pour la kinase Aurora A qui est surexprimée dans plusieurs cancers comme celui du sein, du côlon et l'HCC. Prise dans son ensemble, notre étude montre le rôle potentiel de Merline dans les tumeurs invasives telles que celles rencontrées dans les cancers du sein.