Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.
Bibliographical noteFunding Information:
Address all correspondence to: Dr. Aree Moon, College of Pharmacy, Duksung Women’s University, Seoul 132-714, South Korea. E-mail: email@example.com 1This work was supported by the Korean Science and Engineering Foundation/ National Research Laboratory Program (MEST, no. ROA-2008-000-20070-0) and by the National Research Foundation of Korea (MEST, no. R11-20100001707). 2This article refers to supplementary materials, which are designated by Figures W1 and W2 and are available online at www.neoplasia.com. Received 30 July 2010; Revised 5 November 2010; Accepted 15 November 2010 Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1522-8002/11/$25.00 DOI 10.1593/neo.101088