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 sympathetic 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.

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.

contributed equally to this workThe 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 modi®cations 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 ®ndings constitute a novel example of a reciprocal connection between transcription factors and the cell cycle machinery.

Mutation of the Neurofibromatosis 2 (NF2) tumor suppressor gene leads to cancer development in humans and mice. Recent studies suggest that Nf2 loss also contributes to tumor metastasis. The Nf2-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) 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 of Nf2 deficiency 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 in Nf2 −/− 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.

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 that c-jun mRNA 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.

No abstract

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 ®broblasts 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 Fra1 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 ®broblasts 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.

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 cAMPelevating 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 p44 mapk 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, p44 mapk activates AP-1. Then AP-1 could stimulate tyrosinase expression through the interaction with specific DNA sequences present in the mouse tyrosinase promoter.In melanocytes and melanoma cells, melanin synthesis is controlled by a cascade of enzymatic reactions regulated at the level of tyrosinase. This enzyme synthesizes dopaquinone from tyrosine and appears to control the rate-limiting step of melanogenesis. Melanin synthesis is stimulated by a large array of effectors including 1-oleyl-2-acetyl-glycerol (1), ultraviolet B radiations (2), and cAMP-elevating agents (forskolin, IBMX, 1 ␣-MSH) (3-5). Few data are available concerning the molecular mechanisms triggered by these melanogenic agents. Protein kinase C was thought to be involved in the induction of melanogenesis by 1-oleyl-2-acetyl-glycerol and ultraviolet B radiations (6, 7). However, a recent report of Carsberg et al. (8) has shown that the stimulation of melanogenesis by these agents was not affected by RO485, a potent inhibitor of protein kinase C. While the role of protein kinase C in the induction of melanogenesis remains controversial, compelling data have shown that cAMP-elevating agents stimulate melanogenesis in both melanocytes and melanoma cells, indicating that the cAMP pathway plays a key role in the regulation of melanogenesis (3-5). The effect of cAMP on melanogenesis is due to a stimulation of tyrosinase activity. This appears to be the consequence of an augmentation of enzymatic activity of preexisting tyrosinase (4, 9) following post-translational modifications such as (i) phosphorylation or glycosylation (10), (ii) association with an activator (11, 12), and (iii) dissociation from an inhibitor (13). Alternatively, cAMP was shown to increase tyrosinase mRNA (14, 15), resulting in an augmentation of tyrosinase amount, suggesting that cAMP stimulates tyrosinase transcription (16). However, the molecular events connecting the stimulation of t...