Muscarinic receptors activate Ras through a pathway distinct from that mediated through translocation of the exchange factor mSos1 by receptor tyrosine kinases. Here we report that muscarinic receptors can activate another Ras exchange factor, CDC25Mm, or p140Ras-GRF (refs 5,6). In NIH-3T3 cells expressing subtype 1 human muscarinic receptors (hm1), the agonist carbachol selectively increased the specific activity and phosphorylation state of epitope-tagged Ras-GRF. This stimulation was reversed by protein phosphatase 1 (PP1), and prevented by transducin alpha-subunits. Carbachol treatment of neonatal rat brain explants increasd Ras exchange factor activity and the phosphorylation state of endogenous Ras-GRF. In COS-7 cells, cotransfection of hm1 or hm2 receptors with Ras-GRF conferred carbachol-dependent increases in exchange-factor activity, whereas cotransfection with G-protein beta gamma subunits caused a constitutive activation that was sensitive to PP1. These results demonstrate a G-protein-coupled mechanism for Ras activation, mediated by p140 Ras-GRF.

Activation of mitogen-activated protein kinase (Erk/ MAPK) is a critical signal transduction event for estrogen (E 2 )-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E 2 induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E 2 -induced Erk activation is dependent on new protein synthesis, suggesting that E 2 -induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E 2 in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60 -70% of E 2 -induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E 2 -treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10 -20% of E 2 -induced Erk activation, suggesting that E 2 -induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-␦-mediated signaling by a dominant negative mutant or the relatively specific PKC-␦ inhibitor rottlerin blocked most of the E 2 -induced Erk activation but had no effect on TGF␣-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E 2 -and TGF␣-induced Erk activation. This evaluation of downstream signaling revealed that E 2 -induced Erk activation is mediated by a HRG/HER-2/PKC-␦/Ras pathway that could be crucial for E 2 -dependent growth-promoting effects in early stages of tumor progression.

Ras oncoproteins play pivotal roles in both the development and maintenance of many tumor types. Unfortunately, these proteins are difficult to directly target using traditional pharmacological strategies, in part due to their lack of obvious binding pockets or allosteric sites. This obstacle has driven a considerable amount of research into pursuing alternative ways to effectively inhibit Ras, examples of which include inducing mislocalization to prevent Ras maturation and inactivating downstream proteins in Ras-driven signaling pathways. Ras proteins are archetypes of a superfamily of small GTPases that play specific roles in the regulation of many cellular processes, including vesicle trafficking, nuclear transport, cytoskeletal rearrangement, and cell cycle progression. Several other superfamily members have also been linked to the control of normal and cancer cell growth and survival. For example, Rap1 has high sequence similarity to Ras, has overlapping binding partners, and has been demonstrated to both oppose and mimic Ras-driven cancer phenotypes. Rap1 plays an important role in cell adhesion and integrin function in a variety of cell types. Mechanistically, Ras and Rap1 cooperate to initiate and sustain ERK signaling, which is activated in many malignancies and is the target of successful therapeutics. Here we review the role activated Rap1 in ERK signaling and other downstream pathways to promote invasion and cell migration and metastasis in various cancer types.

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co-stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas-induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf-1/MAPK pathway versus JNK activation by Fas.