Role of Rap Signaling in the Regulation of ERK Activation and Cell-Cell Adhesion

Rap is a member of the Ras family of small GTPases. Mammalian Rap proteins regulate many biological processes including cell and matrix adhesion, migration, proliferation, cytoskeletal dynamics, cell polarity and secretion. Rap1 is activated by TSH and cAMP in thyroid cells independently of PKA. TSH regulates differentiated gene expression and proliferation. Many of these effects are mediated through PKA. There is disagreement as to whether cAMP signaling through Rap contributes to any of the cellular effects of TSH. Given that thyroid cells express high levels of B-Raf and that Rap1 activates ERK through B-Raf in many cells, we investigated whether ERK activation by TSH is mediated through Epac/Rap. We found that ERK activation by TSH was Rap-independent. Instead, TSH stimulates ERK activity through PKA, Ras, B-Raf and MEK in Wistar rat thyroid cells. Intriguingly, silencing B-Raf impaired TSH-stimulated DNA synthesis. This is the first report to identify B-Raf as a target of TSH and a contributor to TSH-mediated proliferation. The contribution of Epac/Rap to the effects of TSH in thyroid cells remains uncertain. Rap1GAP, an important cellular regulator of Rap activity, is downregulated in many human tumors including melanomas, thyroid, prostate, pancreatic, breast and colon carcinomas. In some instances, Rap1GAP expression was further decreased with tumor progression, suggesting that depletion of Rap1GAP contributes to tumor progression. For this reason the consequences of restoring Rap1GAP expression in human tumor cells were investigated. Given the well-defined role of Rap in the regulation of cell adhesion, we examined the functional consequences of restoring Rap1GAP expression on cell-matrix and cell-cell adhesion in human colon cancer cell lines. Modest overexpression of Rap1GAP sufficient to abolish Rap activity impaired cellmatrix interaction, including cell spreading and the formation of focal adhesions. Moreover, restoring Rap1GAP inhibited the ability of cells to migrate to serum. Under the same conditions, Rap1GAP had no effect on the formation or maintenance of cell-cell contacts. This suggests that Rap1GAP is a more efficient inhibitor of Rap signaling to the extracellular matrix than to cell-cell adhesion. The mechanism underlying the specificity of the inhibitory effects of Rap1GAP on Rap signaling remain to be elucidated. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Judy Meinkoth,PhD