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Larry A. Feig, Ph.D. Director, Biochemistry Graduate Program Professor, Tufts Department of Biochemistry
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Links:ResearchLab MembersRecent Publications |
Research Summary:We study the role of the Ras family of GTPases in both the development of CANCER and in mediating CALCIUM SIGNALING in neurons involved in learning and memory. Studies on Ras proteins in CANCER use both engineered tissue models of human carcinoma as well as animal studies to reveal how Ras influences tumor formation through one of its effector proteins the Ral GTPases (see 2 in Fig. below). We focus on how Ral GTPases contribute to tumor progression in both neoplastic epithelial cells, and supporting stromal cells of these tissues. Studies on Ras proteins in neurons focuses on how Ras-GRF proteins (see 1 in Fig. below) mediate calcium induced synaptic plasticity in the hippocampus that is involved in learning and memory. We also investigate the epigenetic mechanisms underlying how the degree of stimulation in the environment in which an animal lives has long-term and even transgenerational effects on the signaling networks in the brain that mediate synaptic plasticity. |
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Constitutively activated Ras proteins are frequently found in many forms of human cancer, including colon, pancreatic and epidermal squamous cell carcinoma. How they promote oncogenesis through stimulation of their multiple effector proteins (Raf, PI3-K, Ral-GEFS, and others) in each of these cancers remains poorly understood. Evidence from various model systems of cancer supports the idea that Ral-GEFs and their downstream targets, RalA and RalB, can play significant positive roles in Ras-mediated oncogenesis. However, there are hints that RalA and/or RalB may sometimes suppress specific phenomena associated with transformation.
The overall goal of our work is to reveal how the Ral signaling cascade contributes to cancer in both tumor cells and adjacent stromal fibroblasts. In order to understand how Ral GTPases functions in squamous carcinoma of the skin, where activated Ras is often an important component, we used a bioengineered human tissue model human skin. This system allows us to manipulate the Ral signaling cascade in either epithelial cells (HaCaT keratinocytes) of the epidermis or fibroblasts of the dermis. This work is done in collaboration with the laboratory of Dr. Jonathan Garlick who runs the Tufts Center for Integrated Tissue Engineering, http://dental.tufts.edu/CITE/. In these tissues, expression on oncogenic 12V-Ras (II-4 cells) is not sufficient to induce invasive behavior. Invasiveness is observed if E-cadherin function is also inhibited by expression of a dominant negative E-cadherin (II-4-dnE-cad).
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Future goals:
A) Ral proteins, RalA and RalB, are known to regulate the exocyst, a protein complex involved in vesicle sorting. As such, Ral proteins may influence polarity of epithelial cells to influence Ras-mediated tumorigenesis. Our preliminary data, soon to be published, show that in keratinocytes RalA actually SUPPRESSES, rather than promotes, Ras-induced tumorigenesis. This is most likely due to the ability of RalA, but not RalB, to stimulate exocyst function and enhance delivery of the tumor suppressor E-cadherin to the basolateral surface of epithelial cells.
In fact, we found that knockdown of RalA, but not RalB, expression with shRNA in this system enhances Ras-induced tumorigenesis when these tissues are transplanted onto the back of mice (see below). Studies on the mechanism involved, and whether this role for RalA applies to other tumor system are in progress.
B) Ral proteins may have distinct exocyst-mediated functions in stromal fibroblasts by influencing secretion of factors that influence tumor progression of neighboring epithelial cells. Studies on the consequence of blocking Ral function in stromal fibroblasts in these engineered tissues are under way.
Ras-GRF1 and Ras-GRF2 are two closely related exchange factors expressed highly in the brain that have the capacity to activate both Ras and Rac in response to calcium influx into cells. Despite their similar overall structure Ras-GRF1 and Ras-GRF2 mediate opposing forms of synaptic plasticity in the highly investigated CA1 region of the hippocampus that are known to contribute to learning and memory. GRF1 mediates NMDA receptor-induced LTD, while GRF2 mediates NMDA receptor induced LTP. At least part of this difference is due to the fact that although both proteins can activate both Ras and Rac effectors, GRF1 predominantly activates p38 MAP kinase, while GRF2 predominantly mediates Erk MAP kinase. A hint at the mechanism underlying this difference is our observation that the two exchange factors mediate the action of different sub-classes of NMDA receptors. GRF1 and GRF2 also make distinct contributions to hippocampus-mediated learning and memory.
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Future goals:
A) We are performing/structure function analysis of GRF1/GRF2 chimeras to test the hypothesis that specific NMDA receptor subtypes drive signaling output from GRF proteins.
B) Both subtypes of NMDA receptors can promote LTP. Yet GRFs only mediate LTP driven by NR2A containing NMDA receptors. It is known that the ratio of NR2A to NR2B receptors changes upon experience. Thus, we are testing the hypothesis that GRF2 generates a unique form of LTP emanating from NR2A receptors through its ability to activate a distinct set of downstream signaling pathways.
C) GRF1 and GRF2 only become important for synaptic plasticity upon entry into adolescence when the hippocampus first becomes important for learning and memory. Before that the Sos exchange factor regulates NMDA receptor activation of Ras. Thus, we plan to test the hypothesis that GRF proteins activate distinct signaling pathways downstream from Ras and Rac in the differentiated hippocampus function due to its interaction with distinct scaffold proteins.
We discovered that the signaling network responsible for promoting LTP in the hippocampus of mice is modulated by the degree of stimulation in their environment. In particular, exposure of adolescent but not adult mice to 2-weeks of an enriched environment unlocks an otherwise latent NMDA receptor/p38 Map kinase signaling cascade (see Fig. below) that contributes to LTP induction. Moreover, this environmentally induced signaling cascade can compensate for the loss of Erk signaling that occurs when GRF2 is lacking in mice. Interestingly, a similar phenomenon does not occur in adult mice. (See Fig. below and Li et al Current Biology 2006).
This phenomenon lasts for at least 3 months in enriched juvenile mice, whereupon it wanes and is absent by 6 months of age. Remarkably, this effect is passed on to the offspring of these enriched mice (see Fig. below and Arai et al Journal of Neuroscience 2009)
Future Goals:
A) We are attempting to define how p38 Map kinase promotes LTP in this environmentally-induced signaling pathway using a phospho-proteomic approach.
B) We are attempting to define how exposure to an enriched environment unlocks the NMDA receptor/p38 signaling cascade in young animals. We assume that an epigenetic change in expression of a rate-limiting step in the signaling pathway is involved.
C) To define how this phenomenon is passed on to offspring of enriched adolescent mice, high-throughput genome-wide screening for EE-induced DNA methylation sites on genes in offspring of enriched mice is being performed in collaboration with the Alex Meissner’s lab at Harvard. An alternative genome-wide approach to identify genes responsible for this phenomenon will be to perform high-throughput profiling of histone modifications in DNA.
For more details, please see the publications below.
Li, S., Tian, X., Hartley, D.M. and Feig, L.A. 2006. The environment versus genetics in controlling the contribution of MAP kinases to synaptic plasticity. Current Biology. 16:1-11.
Tian, X., and Feig, L.A. 2006. “Age-dependent Participation of Ras-GRF Proteins in Coupling Calcium-permeable AMPA Glutamate Receptors to Ras/Erk Signaling in Cortical Neurons.” Journal of Biological Chemistry 281(11): 7578-7582
Yu, Y., Hao, Y, and Feig, L.A. 2006. The R-Ras GTPase mediates cross talk between estrogen and insulin signaling in breast cancer cells. Mol. Cell. Biol. 26:6372-6380.
Li, Shaomin; Tian, Xuejun; Hartley, Dean M; Feig, Larry A. (2006). “Distinct Roles for Ras-Guanine Nucleotide-Releasing Factor 1 (Ras-GRF1) and Ras-GRF2 in the Induction of Long-Term Potentiation and Long-Term Depression.” Journal of Neuroscience 26(6): 1721-1729
Feig, Larry A. (2006). “The Odyssey of K-Ras.” Molecular Cell 21(4): 447-449
Wei, Jie; Fain, Sebastian; Harrison, Celia; Feig, Larry A; Baleja, James D. (2006). “Molecular Dissection of Rab11 Binding from Coiled-Coil Formation in the Rab11-FIP2 C-Terminal Domain.” Journal of the American Chemical Society, in press.
Lim Kian-Huat, Baines, A., Fiordalisi, J.J, Shipitsin, M., Feig, L.A., Cox, A.D., Der, C.J., Counter, C.M. 2005. Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell. 7:533-545.
Rusanescu, G., Yeng, W. Bai, A., Neel, B.G. and Feig, L.A. 2005. Tyrosine phosphatase SHP-2 is a mediator of activity dependent neuronal excitotoxicity. EMBO J. 24:1-10.
Shipitsin, M. and Feig, L.A. 2004. RalA but not RalB enhances polarized membrane delivery of E-cadherin to the basolateral surface of MDCK cells. Mol. Cell. Biol. 24:5746-5756.
Tian, X, Gotoh, T., Tsuji, K, Lo, E., Su, H. and Feig, L.A. 2004 Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras/Erk/CREB. EMBO J. 23:1567-1575.
Feig, L.A., 2003, The Ral GTPases: Approaching their 15 minutes of fame. Trends in Cell Biology 13:419-425.
Buchbaum, R.J., Connoly, B.A. and Feig, L.A. 2003 Regulation of p70 S6 kinase by complex formation between the Rac GEF Tiam1 and the scaffold spinophilin. J. Biol. Chem. 278: 18833-18841.
Polzin A, Shipitsin M, Goi T, Feig LA, Turner TJ., 2002 Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles. Mol Cell Biol. 22:1714-22.
Yu, Y. and Feig, L.A.2002 Involvement of R-Ras and Ral GTPases in estrogen-independent proliferation of cancer cells . Oncogene. 21:7557-756831.
Tian X, Rusanescu G, Hou W, Schaffhausen B, and Feig LA., 2002. PDK1 mediates growth factor-induced Ral-GEF activation by a kinase-independent mechanism EMBO J. 21:1327-38.
Feig, L.A. and Buchsbaum, R. 2002 Cell Signaling: Life or death decisions of Ras proteins Current Biology 12:259-61.
Goi, T., Shipistin, M., Lu, Z., Foster D.A., Klinz, S. and Feig, L.A. 2000, An EGF receptor/Ral-GTPase signaling regulates c-Src activity and substrate specificity. EMBO J. 19, 623-630.
Farnsworth, C.L., Freshney, N.W., Rosen, L.B., Ghosh, A., Greenberg, M.E. and Feig, L.A. 1995 calcium activation of Ras mediated by neuronal exchange factor Ras-GRF, Nature, 376, 524-527.
Jiang, H., Luo, J.-Q., Urano, T., Frankel, P., Lu, Z., Foster, D.A., and Feig, L.A. 1995 Involvement of Ral GTPase in v-Src-induced phospholipase D activation. Nature, 378, 409-412.
