Summary Hyperactivation of Ras-ERK1/2 signaling is critical to the development of many human malignancies but little is known regarding the specific contribution of ERK1 or ERK2 to oncogenic processes. We demonstrate that ERK2 but not ERK1 signaling is necessary for Ras-induced epithelial-to-mesenchymal transformation (EMT). Further, ERK2 but not ERK1 overexpression is sufficient to induce EMT. Many ERK1/2 interacting proteins contain amino acid motifs, e.g. DEF or D-motifs, which regulate docking with ERK1/2. Remarkably, ERK2 signaling to DEF motif-containing targets is required to induce EMT and correlates with increased migration, invasion and survival. Importantly, the late response gene product Fra1 is necessary for Ras- and ERK2-induced EMT through up-regulation of ZEB1/2 proteins. Thus, an apparent critical role for ERK2 DEF motif signaling during tumorigenesis is the regulation of Fra1, and the subsequent induction of ZEB1/2, suggesting a potential therapeutic target for Ras-regulated tumorigenesis.

8p11 myeloproliferative syndrome (EMS) is a hematopoietic stem cell disorder characterized by myeloid hyperplasia and non-Hodgkin's lymphoma with chromosomal translocations fusing several genes, most commonly ZNF198, to fibroblast growth factor receptor-1 (FGFR1). However, patients with BCR-FGFR1 fusion present with typical chronic myeloid leukemia (CML). We demonstrate that ZNF198-FGFR1 induces EMS-like disease in mice, with myeloproliferation and T lymphoma arising from common multipotential progenitors. Mutation of FGFR1 Tyr766 attenuates both myeloid and lymphoid diseases, identifying phospholipase C-gamma1 as a downstream effector. Bcr-FGFR1 binds Grb2 via Bcr Tyr177 and induces CML-like leukemia in mice, whereas Bcr-FGFR1/Y177F lacks Grb2 binding and causes EMS-like disease. These results implicate different signaling pathways originating from both kinase and fusion partner in the pathogenesis of CML and EMS.

Inhibitors of the oncogenic Ras-MAPK pathway have been intensely pursued as therapeutics. Targeting this pathway, however, presents challenges due to the essential role of MAPK in homeostatic functions. The phosphorylation and activation of MAPK substrates is regulated by protein-protein interactions with MAPK docking sites. Active ERK1/2 (extracellular signal-regulated kinase 1/2)-MAPKs localize to effectors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P)- or D-domain (docking domain) motifs. We have examined the in vivo activity of ERK2 mutants with impaired ability to signal via either docking site. Mutations in the DEF-domain binding pocket prevent activation of DEF-domain-containing effectors but not RSK (90 kDa ribosomal S6 kinase), which contains a D domain. Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RSK activation but not DEF-domain signaling. Uncoupling docking interactions does not compromise ERK2 phosphotransferase activity. ERK2 DEF mutants undergo regulated nuclear translocation but are defective for Elk-1/TCF transactivation and target gene induction. Thus, downstream branches of ERK2 signaling can be selectively inhibited without blocking total pathway activity. Significantly, several protooncogenes contain DEF domains and are regulated by ERK1/2. Therefore, disrupting ERK-DEF domain interactions could be an alternative to inhibiting oncogenic Ras-MAPK signaling.

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-ornothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.Ultrasensitive switch-like responses control cell fate decisions in many biological settings, and the regulation of kinase activity is one way in which ultrasensitive behavior can be initiated (9,14). Such behavior is characterized by an all-ornothing response to increasing concentrations of stimulus (Fig. 1A). An ultrasensitive switch-like response displays a near vertical sigmoidal stimulus response curve. In the case of extreme ultrasensitive responses, signaling molecules switch between two discontinuous stable states and intermediate responses are not observed; this is referred to as a bistable response. Bistability may result from multistep phosphorylation events, positive feedback loops, double-negative feedback loops, and the relative abundance/activity of kinases and phosphatases driving the signaling cascade. In contrast to ultrasensitive signaling, a graded response in protein kinase activity changes incrementally with the stimulus concentration (Fig. 1B). Given the irreversible, all-or-nothing nature of many cell behaviors, including differentiation and cell cycle control, significant efforts have been focused on identifying the cellular mechanisms underlying bistability in biological systems.Bistability in mitogen-activated kinase (MAPK) signaling was first demonstrated experimentally by the progesteroneinduced maturation of Xenopus laevis oocytes. In pools of oocytes incubated with increasing concentrations of progesterone, extracellular signal-regulated kinase (ERK) activity appears to increase in a graded manner. However, whe...

HIV-1 associated dementia is thought to be caused by neuronal damage and death in response to the production of soluble neurotoxic factors by virally infected mononuclear phagocytes. These neurotoxins include HIV-1 Tat. The ability of neurotrophins to promote cell survival prompted us to examine whether neurotrophins might also be capable of opposing the pro-apoptotic effects of Tat. Here, we show that Tat-induced neuronal apoptosis in primary cultures of rat cerebellar granule cells and in neuronally differentiated human SK-N-MC cells is profoundly inhibited by brain-derived neurotrophic factor, nerve growth factor and activity-dependent neurotrophic factor nonamer peptide. These neurotrophins activated the transcription factor NF-kB, and inhibition of NF-kB activation using a super-repressor IkB-a mutant was found to block the survival-promoting activity of the neurotrophins. Reporter gene assays and immunoblot experiments revealed that the neurotrophins also up-regulated the expression of Bcl-2, at both the transcriptional and protein levels. Overexpression of the super-repressor IkB-a mutant prevented this induction of Bcl-2 expression. Moreover, overexpression of either Bcl-2, alone, or the RelA subunit of NF-kB, alone, protected neurons from Tat-induced apoptosis. These ®ndings suggest that the activation of NF-kB by neurotrophic factors may promote survival of neurons exposed to Tat, via regulation of anti-apoptotic genes including Bcl-2.