Adhesion Dependent Regulatory Crosstalk Of Small GTPases Ral and Arf6 and its Role in Anchorage Dependent Signaling

Abstract

Anchorage independent growth of cancer cells is a key component of cancer invasion and metastasis. Cancer metastasis is a major cause of mortality, making the understanding of the basis for anchorage dependence and how it is overcome in cancers an important problem. Oncogenic Ras induced transformation in cancers drives anchorage independence through activation of small GTPases of the Ral family, RalA and RalB. Our previous studies in mouse embryonic fibroblasts (MEFs) have shown integrin mediated cell adhesion to the extra cellular matrix activates RalA (not RalB). Active RalA mediates the exocyst-dependent trafficking of membrane raft microdomains to the plasma membrane, to stimulate adhesion dependent signaling. Constitutive activation of RalA downstream of oncogenic Ras in cancers uses this pathway to support anchorage independent signaling. Like RalA, the small GTPase Arf6 is also activated by integrin mediated adhesion and regulates this raft trafficking pathway. Unlike RalA however Arf6 is necessary but not sufficient for this trafficking. Interestingly, RalA and Arf6 are reported to regulate many common cellular functions including GLUT4 receptor recycling, insulin secretion and cytokinesis, sharing signaling partners such as exocyst complex, RalBP1 and Phospholipase D1.

This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T-24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T-24 cells the Ral-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Ral and Arf6 co-precipitate with each other in a functional complex that could mediate this crosstalk. We hence investigated the role of Ral effector proteins, Sec5, Sec10, Exo84, RalBP1 and Phospholipase D1, in mediating Arf6 activation and found them to differentially regulate Arf6. Active Ral uses Ral-RalBP1-ARNO-Arf6 pathway in T-24 cells and MEFs to mediate Arf6 activation. We also looked at the role of GEF proteins in differential activation of RalA/RalB in normal and cancer cells. Our studies identify a novel role for the Ral GEF, RGL1, in mediating the differential activation of RalA downstream of integrins in MEFs and oncogenic Ras in colorectal cancer cell line SW620.

In summary, this study has identified and evaluated a novel regulatory crosstalk between Ral and Arf6, testing its functional significance in mediating anchorage independent signaling and growth. It has also identified a novel role for the RalGEF RGL1 in mediating the integrin dependent differential activation of RalA. In doing so it has revealed a new understanding of how Ral isoforms (RalA and RalB) are differentially regulated and in turn regulate downstream signaling in normal and cancers.