Cavin-3 Dictates the Balance Between ERK and Akt Signaling
ABSTRACT: Cavin-3 is a tumor suppressor protein of unknown function. Using a combination of in vivo knockout and in vitro gain/loss of function approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae, a lipid-raft specialization that contains an ERK activation module, to the membrane skeleton of the plasma membrane. Loss of cavin-3 reduces the number of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation and resistance to apoptosis. The in vivo consequences of cavin-3 loss are increased lactate production and cachexia. 9 total samples, consisting of 3 cavin-3 siRNA groups (0 days, 3 days and 8 days) one set was untreated, one set was serum starved, one set was serum starved and then treated with EGF for 1 hr.
Project description:Cavin-3 is a tumor suppressor protein of unknown function. Using both in vivo and in vitro approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae to the membrane skeleton of the plasma membrane via myosin-1c. Caveolae are lipid raft specializations that contain an ERK activation module and loss of the cavin-3 linkage reduces the abundance of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation, and resistance to apoptosis. The in vivo consequences of cavin-3 knockout are increased lactate production and cachexia. DOI:http://dx.doi.org/10.7554/eLife.00905.001.
Project description:Tumor cells are capable of surviving loss of nutrients and anchorage in hostile microenvironments. Under these conditions, adapting to specific signaling pathways may shift the balance between growth and cellular dormancy. Here, we report a mechanism by which epidermal growth factor receptor (EGFR) differentially modulates the phosphatidylinositol 3'-kinase (PI3K)/AKT pathway in cellular stress conditions. When carcinoma cells were cultured as multicellular aggregates (MCA), cyclin D1 was induced through a serum-dependent EGFR activating pathway, triggering cell proliferation. The expression of cyclin D1 required both EGFR-mediated ERK and AKT activation. In serum-starved MCAs, EGFR activation was associated with active ERK1/2, but not AKT, and failed to induce cyclin D1. Analysis revealed that, under serum-starved conditions, EGFR-Y1086 residue was poorly autophosphorylated and this correlated with failure to phosphorylate Gab1. Accordingly, the EGFR activation failed to induce EGFR/PI3K complex formation or AKT activation, preventing cyclin D1 induction. Furthermore, we show that in serum-starved MCA, expression of constitutively active AKT re-established cyclin D1 expression and induced proliferation in an EGFR-dependent manner. Thus, modulation of the PI3K/AKT pathway by context-dependent EGFR signaling may regulate tumor cell growth and dormancy.
Project description:The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular therapeutic dilemma, as direct targeting of Ras proteins by small molecules has proved difficult. Signaling pathways downstream of Ras, in particular Raf/Mek/Erk and PI3K/Akt/mTOR, are dominated by lipid and protein kinases that provide attractive alternate targets in Ras-driven tumors. As p21-activated kinase 1 (Pak1) has been shown to regulate both these signaling pathways and is itself upregulated in many human cancers, we assessed the role of Pak1 in Ras-driven skin cancer. In human squamous cell carcinoma (SCC), we found a strong positive correlation between advanced stage and grade and PAK1 expression. Using a mouse model of Kras-driven SCC, we showed that deletion of the mouse Pak1 gene led to markedly decreased tumorigenesis and progression, accompanied by near total loss of Erk and Akt activity. Treatment of Kras(G12D) mice with either of two distinct small molecule Pak inhibitors (PF3758309 and FRAX597) caused tumor regression and loss of Erk and Akt activity. Tumor regression was also seen in mice treated with a specific Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as a new target in KRAS-driven tumors and suggest a mechanism of action through the Erk, but not the Akt, signaling pathway.
Project description:Huntington's disease (HD) is a fatal inherited neurodegenerative disorder. HD is caused by polyglutamine expansions in the huntingtin (htt) protein that result in neuronal loss and contribute to HD pathology. The mechanisms of neuronal loss in HD are elusive, and there is no therapy to alleviate HD. To find small molecules that slow neuronal loss in HD, we screened 1,040 biologically active molecules to identify suppressors of cell death in a neuronal cell culture model of HD. We found that inhibitors of mitochondrial function or glycolysis rescued cell death in this cell culture and in in vivo HD models. These inhibitors prevented cell death by activating prosurvival ERK and AKT signaling but without altering cellular ATP levels. ERK and AKT inhibition through the use of specific chemical inhibitors abrogated the rescue, whereas their activation through the use of growth factors rescued cell death, suggesting that this activation could explain the protective effect of metabolic inhibitors. Both ERK and AKT signaling are disrupted in HD, and activating these pathways is protective in several HD models. Our results reveal a mechanism for activating prosurvival signaling that could be exploited for treating HD and possibly other neurodegenerative disorders.
Project description:A multitude of factors regulate oligodendrocyte differentiation and remyelination, and to elucidate the mechanisms underlying this process, we analyzed the interactions of known signaling pathways involved in these processes. Previous work from our lab and others shows that Akt, mTOR, and Erk 1/2 are major signaling pathways regulating oligodendrocyte differentiation and myelination in vitro and in vivo. However, the relative contribution of the different pathways has been difficult to establish because the impact of inhibiting one pathway in in vitro cell culture models or in vivo may alter signaling through the other pathway. These studies were undertaken to clarify the interactions between these major pathways and understand more specifically the crosstalk between them. Oligodendrocyte differentiation in vitro required Akt, mTOR, and Erk 1/2 signaling, as inhibition of Akt, mTOR, or Erk 1/2 resulted in a significant decrease of myelin basic protein mRNA and protein expression. Interestingly, while inhibition of the Erk1/2 pathway had little impact on Akt/mTOR signaling, inhibition of the Akt/mTOR pathways significantly increased Erk1/2 signaling, although not enough to overcome the loss of Akt/mTOR signaling in the regulation of oligodendrocyte differentiation. Furthermore, such crosstalk was also noted in an in vivo context, after mTOR inhibition by rapamycin treatment of perinatal pups. GLIA 2014;62:2096-2109.
Project description:The RAS/ERK and PI3K/AKT pathways induce oncogenic gene expression programs and are commonly activated together in cancer cells. Often, RAS/ERK signaling is activated by mutation of the RAS or RAF oncogenes, and PI3K/AKT is activated by loss of the tumor suppressor PTEN. In prostate cancer, PTEN deletions are common, but, unlike other carcinomas, RAS and RAF mutations are rare. We have previously shown that over-expression of "oncogenic" ETS transcription factors, which occurs in about one-half of prostate tumors due to chromosome rearrangement, can bypass the need for RAS/ERK signaling in the activation of a cell migration gene expression program. In this study we test the role of RAS/ERK and PI3K/AKT signaling in the function of oncogenic ETS proteins.We find that oncogenic ETS expression negatively correlates with RAS and RAF mutations in prostate tumors. Furthermore, the oncogenic ETS transcription factors only increased cell migration in the absence of RAS/ERK activation. In contrast to RAS/ERK, it has been reported that oncogenic ETS expression positively correlates with PI3K/AKT activation. We identified a mechanistic explanation for this finding by showing that oncogenic ETS proteins required AKT signaling to activate a cell migration gene expression program through ETS/AP-1 binding sequences. Levels of pAKT correlated with the ability of oncogenic ETS proteins to increase cell migration, but this process did not require mTORC1.Our findings indicate that oncogenic ETS rearrangements cause a cell migration gene expression program to switch from RAS/ERK control to PI3K/AKT control and provide a possible explanation for the high frequency of PTEN, but not RAS/RAF mutations in prostate cancer.
Project description:Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.
Project description:Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein-coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)-dependent G(1)-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G(1)-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a G(i)-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through G(i), anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of G(i) and unaffected by anastellin. These results suggest that lysophospholipid mediators of G(i) activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of G(i) and that anastellin selectively inhibits the Ras/ERK arm of the pathway.
Project description:CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.