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"REGULATION OF VESICLE TRANSPORT AND CELL MOTILITY BY GOLGI-LOCALIZED DBS"

by
Ethan Fitzpatrick
Interdisciplinary Biomedical Sciences Program
B.S. 2002, Rider University



Thesis Advisor: Ian Whitehead, Ph.D.
Professor
Department of Microbiology & Molecular Genetics

Tuesday, August 3, 2010
Cancer Center, G Level Seminar Room, 9:00 A.M.


Abstract

Dbs was first identified in a screen for cDNAs that, when over expressed, cause deregulated growth in a fibroblast cell line. Since then the molecular basis for this transformation has been studied extensively. It was determined that oncogenic Dbs induced transformation results from the aberrant activation of RhoA, resulting in the downstream phosphorylation of myosin light chain through the effector ROCKI. Recent work has been performed using more complete Dbs cDNAs than the one originally identified that contain various homology domains outside the Rho GEF catalytic domain of oncogenic Dbs. Results of studies where over expressed full length Dbs was used have shown that the various domains found in the full length protein can contribute to the proteins function. Dbs has been identified as a regulator of various biological processes such as motility and endocyosis in several cell types. Furthermore, transcriptional isoforms of Dbs have been identified that differ in the regulatory domains outside the RhoGEF domain. In the current study we have identified Dbs isoforms at the protein level and found that these isoforms can localize to the discrete sub cellular compartments of the Golgi apparatus and Endoplasmic Reticulum. We have determined that the Sec14 domain of Dbs can be used to target the full length protein to the Golgi and that the isolated Sec14 domain retains this Golgi targeting function. The Sec14 domain was found to bind to the catalytic DH domain responsible for Rho GEF activity and could be used to inhibit exchange activity of the endogenous protein. These unique properties of the Dbs Sec14 domain (i.e. self targeting to the Golgi and inhibition of exchange activity) allowed us to use the over expressed Sec14 domain as an inhibitor of Golgi localized endogenous Dbs. Thus, the Sec14 domain inhibitor was used to dissect the function of the endogenous protein. Our results indicate that Golgi localized Dbs is involved in the regulation of membrane dynamics at the Golgi and that this function is involved in supporting motility in a metastatic cancer cell line. This data suggests that Dbs may be a potential therapeutic target to inhibit the metastatic properties of cancer cells and also that intra molecular inhibitory domains may, more generally, be used as a tool for studying the function of endogenous proteins.


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