FUNCTION OF BPGAPI-SMGGDS INTERACTION IN CELL SIGNALING

Abstract

BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain containing, Proline-rich and Cdc42GAP-like protein subtype-1), is a ubiquitously expressed, RhoGAP. It is a multi-domain protein that regulates the formation of pseudopodia and cell migration via the interactions of its BCH, GTPase-activating (GAP) and proline-rich domains. Through the binding of cortactin, an actin-binding protein, to the proline-rich motif, BPGAP1 enhances cell migration whereas the binding of EEN/endophilin2 to the same proline-rich motif enhances Ras/MAPK pathway activation via EGF receptor endocytosis. The BCH domain of BPGAP1 has been found to be multifunctional. It can enhance the Ras/MAPK pathway independent of EEN-mediated EGF receptor endocytosis. The BCH domain is responsible for homophilic and heterophilic interaction with other BCH-domain containing proteins. It was previously shown that SmgGDS (Small G-protein GDP Dissociation Stimulator) could interact with both the BCH domain of BPGAP1 and the small GTPase, K-Ras. The significance of such interactions however remained unknown.

Here we further examine the interactions among SmgGDS, K-Ras and the BCH domain and their role in regulating differentiation. We have shown that the BCH domain-mediated activation of ERK1/2 and subsequent protrusion formation in PC12 cells takes place via the Ras/MAPK pathway and the activation of K-Ras. Knockdown of SmgGDS revealed a negative regulation towards BCH domain-mediated activation of K-Ras and protrusion formation in PC12 cells. Further deletion studies show that the BCH domain and K-Ras bind to different regions on SmgGDS. Disruption of binding of either K-Ras or the BCH domain to SmgGDS does not release the blocking of BCH domain-mediated protrusions in PC12 cells. Interaction of endogenous SmgGDS with BPGAP1 is weaker than its interaction with the BCH domain. We have also discovered the existence of novel intramolecular interaction sites within BPGAP1 that could modulate SmgGDS interaction.

In conclusion, the BCH domain of BPGAP1 causes protrusions upon stimulation of PC12 cells by activating K-Ras via the Ras/MAPK pathway, probably by displacing the inhibition by SmgGDS. This thesis work has also unraveled a previously unexpected role of SmgGDS as a negative regulator of K-Ras signaling. The significance of these findings is discussed.