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 precipi