Regulation of Exocyst Function by the Small GTPase RalA.
Abstract: The exocyst
is a multisubunit complex that is required for the vesicular trafficking of many
proteins. Regulation of exocyst assembly occurs in part through multiple small GTPases,
including RalA. Previous work in our lab has shown RalA to be essential for two exocyst
dependent processes, delivery of E-Cadherin to the basolateral membrane of H-Ras
transformed keratinocytes, and the ... read moresecretion of HGF from primary fibroblasts. Some, but
not all, exocyst subunits may be required for specific processes. We have identified
Sec8 as an important regulator of E-Cadherin delivery. Depletion of Sec8 from
keratinocytes grown in organotypic models of squamous cell carcinoma resulted in
decreased ECadherin levels and increased invasion of the keratinocytes into the
underlying dermis. These results phenocopied depletion of RalA implying that Sec8 is
downstream of RalA. In order to gain further insight into the mechanism by which RalA
and Sec8 regulate E-Cadherin delivery we isolated the exocyst complex from control and
RalA depleted tissues by Sec8 immunoprecipitation of the epithelial layer and analyzed
samples by 2D-DIGE. Similar experiments were conducted in fibroblasts to examine the
role of RalA and Sec8 in HGF secretion. While 2D-DIGE failed to identify changes in
proteins associated with Sec8 which may regulate vesicular trafficking of E-Cadherin in
keratinocytes, we were able to identify RPLP1 as a potential Sec8 interacting protein in
fibroblasts. Further work is required to validate the specificity of this interaction
and its contribution to HGF secretion.
Thesis (M.S.)--Tufts University, 2012.
Submitted to the Dept. of Biochemistry.
Advisors: Larry Feig, and Gail Sonenshein.
Committee: Brian Schaffhausen, and Alexei Degterev.
Keyword: Biochemistry.read less