Abstract 2155: Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma

The oncogenes SRC and ERK1 are frequently activated in a wide variety of human cancer, while the tumor suppressor DLC1, which encodes a Rho-GAP (GTPase activating protein) essential for its tumor suppressor functions, is frequently down-regulated. However, no prior research has mechanistically linked SRC and ERK1 to DLC1. In this study, we report that SRC and ERK1 cooperate to attenuate the Rho-GAP and tumor suppressor functions of DLC1 by a previously undescribed mechanism. We determined the direct phosphorylation of Y701 of DLC1, which lies in the Rho-GAP domain, by the SRC kinase reduces the binding of Rho-GTP (active Rho) to the Rho-GAP domain of DLC1 and abolishes its Rho-GAP activity. In untransformed and lung adenocarcinoma cell lines, endogenous active SRC and DLC1 co-localized at focal adhesions and formed a protein complex in vivo, implying this interaction is physiologically relevant. The phosphorylation of S129 of DLC1, which lies N-terminal to the Rho-GAP domain, by ERK1 enhanced the binding of the SRC SH3 domain to this region of DLC1 and increased the phosphorylation of Y701 by SRC. These changes reduced the Rho-GAP activity of DLC1, increased Rho-GTP in the cell, and attenuated the DLC1 tumor suppressor functions, as measured by cell migration rate, anchorage-independent growth, and tumor formation in nude mice. Consistent with these observations, mutation of Y701 to F701 increased the Rho-GAP and tumor suppressor activities of DLC1 and decreased Rho-GTP and Rho/ROCK/MRLC signaling. Conversely, mutation of Y701 to the phosphomimetic D701 produced a mutant DLC1 with the opposite phenotype, similar to a ‘GAP-dead’ DLC1 mutant. The Rho-GAP domain of DLC1 was necessary and sufficient for the attenuated Rho-GAP activity attributable to Y701 phosphorylation, as the isolated Rho-GAP domain (residues 609-878) with the Y701F and Y701D mutants displayed, respectively, high and low Rho-GAP activities, as in full-length DLC1. In considering the potential relevance of these findings to human tumors, it is important to recognize that while the Rho-GAP activity of DLC1 is necessary for its full tumor suppressor activity, it is not sufficient, as DLC1 binds several ligands that contribute to this function without attenuating its Rho-GAP activity. Thus, it would be predicted that there would be selective pressure for down-regulation of DLC1 expression even in the presence of high SRC activity. Consistent with this hypothesis, the combination high SRC expres