Browsing by Subject "Rho GTPases"
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Item Characterization of metastasis regulators in human breast cancer: implications for tumor suppressor PTEN and the Rho family of small GTPases(2005) Baugher, Paige Jennette; Dharmawardane, SuranganiCancer metastasis is a multi-faceted process requiring the disregualtion of numerous signaling pathways, including those associated with cell adhesion and motility. Recent data indicates strongly that growth at a primary tumor site and growth at a metastatic site differ by the expression and/or context-dependent function of the metastasis regulator, and that a wide variety of signaling pathways are affected. PTEN (phosphatase and tensin homologue deleted on chromosome ten) then becomes an attractive candidate for a metastasis suppressor, based on its ability to negatively regulate numerous pathways involved in cell survival, cell proliferation, and cell motility. Conversely, the Rho family of small GTPases have become attractive candidates as contributors to metastasis. Rho GTPases regulate numerous signaling pathways involved in cell survival, cell proliferation and cell motility, but they function to enhance these processes instead of inhibiting them. Data presented here demonstrates the ability of PTEN to negatively regulate motility in human metastatic breast cancer cells without causing the cells to undergo apoptosis. PTEN is localized in stimulated cells away from the leading edge, which displaces it from sites of active motility signaling and prevents it from inhibiting these processes. Furthermore, ectopic PTEN expression is shown to downregulate phosphoinositol (3,4,5) triphosphate (PIP3), expression. Therefore, PTEN could be acting as a metastasis suppressor in human breast cancer. Data presented here also demonstrate the ability of the Rac subfamily of Rho GTPases to enhance metastatic properties and contribute to metastasis. Increased Rac activity was shown to correlate with increased metastatic potential in a panel of metastatic human breast cancer cell variants. When activated Rac1 or Rac3 was expressed stably in the least metastatic variant, either isoform was found to enhance adhesion, migration, and invasion in vitro, as well as contribute to pulmonary metastasis in the nude mouse model of experimental metastasis. Conversely, when dominant negative Rac1 or Rac3 was expressed in the most metastatic variant, either isoform was found to decrease adhesion, migration, and invasion in vitro, as well as block pulmonary metastasis in vivo. Therefore, Rac1 and/or Rac3 are found to act as metastasis regulators by negatively regulating metastatic human breast cancer progression.Item Cloning, characterization of chTC10, a Rho small GTPase, its regulation by Rel/NF-kappaB family members c-Rel and v-Rel, and its role in v-Rel-mediated transformation of fibroblasts(2003-05) Tong, Shun; Bose, Henry R.v-Rel is the oncogenic member of Rel/NF-κB family of transcription factors and transforms hematopoietic cells and fibroblasts. Differential display was employed to identify target genes that are regulated by v-Rel. By using this technique, one gene whose expression is upregulated by v-Rel in chicken embryonic fibroblasts (CEF) was found to encode chTC10, the chicken homolog of TC10, a member of the Rho small GTPase family. The coding region of chTC10 was cloned and shown to encode a 214 amino acids protein sharing 93% identity with human TC10. chTC10 messenger RNA is mainly expressed in heart, skeletal muscle, spleen, and bursa, and is expressed at relatively lower levels in thymus, fat, midbrain, stomach, small intestine, skin and testis from chicken. The chTC10 mRNA and protein levels were increased in v-Rel- or c-Relexpressing CEF. Elevated amounts of GTP-bound chTC10 were also detected in v-Rel-transformed CEF and c-Rel-expressing CEF. chTC10 induced some morphological changes in CEF that were not as obvious as those in mammalian fibroblasts. chTC10Q76L, when highly expressed in CEF, induced numerous short filopodia and cells became rounder. Both wild type and a gain-of-function mutant (chTC10Q76L) enhanced the anchorage-independent growth ability of CEF. Co-expression of chTC10Q76L or chTC10 with v-Rel greatly enhanced CEF colony formation ability in soft agar. By contrast, expression of the dominant negative mutant (chTC10T32N) decreased the colony formation potential of v-Rel. Furthermore, chTC10Q76L increased the saturation density of CEF and this ability was enhanced by its cooperation with v-Rel. These results indicate that chTC10 contributes to transformation of primary fibroblasts by increasing the colony formation ability in soft agar and by increasing cell saturation density. The induction of chTC10 expression and activity contributes to v-Rel-mediated transformation of CEF. In addition, chTC10Q76L activates c-Jun N-terminal kinase (JNK) in response to serum starvation. JNK activity was greatly induced in v-Reltransformed CEF when cells were grown without extracellular matrix. Coexpression with chTC10T32N greatly decreased this activity, suggesting that chTC10 is important for activation of JNK in v-Rel-transformed CEF, and that chTC10 may induce transformation through the JNK pathway.Item Structural basis of RhoA activation by leukemia-associated RhoGEF(2004) Kristelly, Romana, 1972-; Tesmer, JohnSmall GTPases of the Rho family are regulators of cytoskeletal organization, neuronal morphogenesis, and transcription and are implicated in the development of cancer. The activity of Rho GTPases is dependent on their binding to GDP (inactive) or GTP (active) and is tightly regulated by accessory proteins. RhoGEFs (Rho guanine nucleotide exchange factors) activate Rho GTPases by stabilizing their nucleotide-free form via a DH/PH (Dbl homology/Pleckstrin homology) domains module. Leukemiaassociated RhoGEF (LARG) belongs to a subfamily of RhoGEFs, the RH-RhoGEFs, that also contain an RH (Regulator of G protein signaling homology) domain N-terminal to the DH/PH domains and specifically activate RhoA, and not the two other RhoGTPases, Cdc42 and Rac1. RH-RhoGEFs are coupled to G protein-coupled receptor (GPCR) activation because their RH domains interact with Gα12/13 proteins, which in turn activates their GEF activity. Although the LARG DH domain is sufficient for catalysis the PH domain contributes to nucleotide exchange. To better understand how the LARG PH and RH domains contribute to its activity, and to elucidate the structural determinants of RhoA-specificity, structures of the LARG DH/PH domains alone and in complex with RhoA were determined by x-ray crystallography at 2.1 and 3.2 Å resolution, respectively. To verify the structural findings, mutants were generated and assessed using a fluorescence assay. A novel N-terminal subdomain of the DH domain was discovered, which seems to be important for activity and might be used as a switch to regulate LARG activity. The sequence of this N-terminal extension is conserved throughout the Lbc family of RhoA specific RhoGEFs. PH domain-assisted nucleotide exchange in LARG is dependent on the structural integrity of the junction between the DH and PH domains and not on direct contacts of the PH domain with RhoA. A hydrophobic patch on the PH domain has been discovered, which might be a protein-docking site that is used for regulation of GEF activity by other proteins (e.g Gα13) or other domains within LARG (e.g. RH domain). Fluorescence assays of fragments including the RH domain showed an inhibitory effect of the RH domain in vitro, the structural basis of which will be investigated in future experiments.