Oncogenic inhibition by a deleted in liver cancer gene requires cooperation between tensin binding and Rho-specific GTPase-activating protein activities Xiaolan Qian*, Guorong Li*, Holly K. Asmussen*, Laura Asnaghi*, William C. Vass*, Richard Braverman*, Kenneth M. Yamada†, Nicholas C. Popescu‡, Alex G. Papageorge*, and Douglas R. Lowy*§ *Laboratory of Cellular Oncology and ‡Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and †Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892 Communicated by Ira Pastan, National Institutes of Health, Bethesda, MD, April 2, 2007 (received for review March 1, 2007) The three deleted in liver cancer genes (DLC1–3) encode Rho- The prototypic member, designated DLC1, is localized to GTPase-activating proteins (RhoGAPs) whose expression is fre- chromosome 8p21–22 in a region that is commonly deleted in quently down-regulated or silenced in a variety of human malig- hepatocellular carcinoma (5). Its expression is frequently down- nancies. The RhoGAP activity is required for full DLC-dependent regulated or silenced in various solid tumors and hematologic tumor suppressor activity. Here we report that DLC1 and DLC3 bind malignancies, predominantly by promoter methylation (6–13). to human tensin1 and its chicken homolog. The binding has been Ectopic reexpression in DLC1-deficient cancer cell lines can mapped to the tensin Src homology 2 (SH2) and phosphotyrosine suppress cell proliferation, induce apoptosis, and reduce tumor- binding (PTB) domains at the C terminus of tensin proteins. Distinct igenicity. The RhoGAP activity appears to be required for these DLC1 sequences are required for SH2 and PTB binding. DCL binding antioncogenic activities because RhoGAP-deficient mutants to both domains is constitutive under basal conditions. The SH2 have reduced biological activity. The DLC1 protein is reported binding depends on a tyrosine in DCL1 (Y442) but is phosphoty- to have a peripheral cellular location, and the rat homolog has rosine-independent, a highly unusual feature for SH2 binding. been localized to caveolae and focal adhesions (6, 14, 15). DLC1 competed with the binding of other proteins to the tensin C There are two other closely related genes, DLC2 (or terminus, including ␤3-integrin binding to the PTB domain. Point STARD13), located on chromosome 13q12 (16), and DLC3 (or mutation of a critical tyrosine residue (Y442F) in DLC1 rendered the KIAA0189 or STARD8), located on the X chromosome at q13 protein deficient for binding the tensin SH2 domain and binding (17). They have been less intensively examined, but their ex- full-length tensin. The Y442F protein was diffusely cytoplasmic, in pression is also down-regulated in a spectrum of tumors (11, 17). contrast to the localization of wild-type DLC1 to focal adhesions, All three DLC genes encode proteins that are Ϸ1,100 aa, with but it retained the ability to reduce the intracellular levels of three recognized motifs: (i) a sterile ␣ motif (SAM; other SAM Rho-GTP. The Y442F mutant displayed markedly reduced biological motifs are often implicated in protein–protein interactions), (ii) activity, as did a mutant that was RhoGAP-deficient. The results a RhoGAP catalytic domain, and (iii) a START (STAR-related suggest that DLC1 is a multifunctional protein whose biological lipid transfer) domain. Other START motifs are implicated in activity depends on cooperation between its tensin binding and the association with lipid-rich molecules. RhoGAP activities, although neither activity depends on the other. Although these studies establish the DLC family as tumor suppressor genes, there is relatively little insight into their DLC1 ͉ Src homology 2 and phosphotyrosine binding domains ͉ mechanism of action beyond the requirement for the RapGAP tumor suppressor gene activity. It seems unlikely that this activity by itself accounts for the frequent inactivation of DLC members in tumors because t is widely recognized that a combination of genetic and other RhoGAPs do not appear to be commonly down-regulated Iepigenetic changes in the target cell contribute to cancer in tumors. Furthermore, the three recognized motifs in DLC, development (1). These changes may include the activation of namely, SAM, RhoGAP, and START, together account for Ϸ oncogenes or antiapoptotic genes and the inactivation of tumor 500 of the 1,100 aa, which suggests that other putative key suppressor or proapoptotic genes. One feature of many advanced functions may be carried out by these uncharacterized regions. tumors is the activation of the Rho-GTPases RhoA and RhoC. To examine this possibility, we have carried out a yeast two- These small GTPases, which are Ras-related proteins, may hybrid screen with sequences located between the SAM and contribute to various parameters of abnormal cell growth, RhoGAP domains. One interacting protein identified in this including viability, migration, invasion, proliferation, and an- screen was tensin1, which is a peripheral protein implicated in chorage-independent growth (2–4). Although point mutation in Ras genes frequently accounts for their activation in cancer, such Author contributions: X.Q. and G.L. contributed equally to this work; X.Q., K.M.Y., N.C.P., mutations have not been reported for RhoA and RhoC, which A.G.P., and D.R.L. designed research; X.Q., G.L., H.K.A., L.A., and W.C.V. performed re- appear to be wild type in cancer. As with most other small search; X.Q., G.L., H.K.A., and R.B. contributed new reagents/analytic tools; X.Q., G.L., GTPases, the Rho-GTPases are active when bound to GTP and K.M.Y., and D.R.L. analyzed data; and X.Q. and D.R.L. wrote the paper. inactive when bound to GDP. They can be activated by stimu- The authors declare no conflict of interest. lation of Rho-specific guanine nucleotide exchange factors, Freely available online through the PNAS open access option. which increase the level of Rho-GTP and reduce the level of Abbreviations: SH2, Src homology 2; pY, phosphotyrosine; PTB, pY binding; RhoGAP, Rho-GDP, or inactivated by Rho-specific GTPase-activating Rho-specific GTPase-activating protein; FL, full-length; LPA, lysophosphatidic acid; SAM, ␣ proteins (RhoGAPs), which reduce the level of Rho-GTP and sterile motif; DLC, deleted in liver cancer. §To whom correspondence should be addressed at: Laboratory of Cellular Oncology, increase the level of Rho-GDP. By far the most common National Cancer Institute, National Institutes of Health, Building 37, Room 4106, alteration reported for Rho regulators in cancer is the inactiva- Bethesda, MD 20892. E-mail: [email protected]. tion of RhoGAPs that are members of the deleted in liver cancer This article contains supporting information online at www.pnas.org/cgi/content/full/ (DLC) family. 0703033104/DC1. 9012–9017 ͉ PNAS ͉ May 22, 2007 ͉ vol. 104 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703033104 Downloaded by guest on September 25, 2021 those lacking amino acids 468–476 or 476–490 had binding activity similar to wild-type DLC1 (SI Fig. 8A). SH2 and PTB domains are docking sites for protein–protein interaction (19, 20). Although PTB stands for phosphotyrosine (pY) binding, many PTB domains can bind their ligands in a pY-independent manner, and the PTB domain in tensin is phylogenetically grouped with this latter class of PTB domains (20). By contrast, almost all SH2 domains require pY (19). We therefore initially assumed that the PTB domain of tensin would be primarily responsible for the interaction with DLC1 because the binding to tensin appeared to be pY-independent, given the positive yeast two-hybrid results, where pY is low, and data showing that DLC1 efficiently bound the tensin SH2-PTB region in nontransformed mammalian cells, bound with similar effi- ciently in v-Src-transformed cells, and did not contain detectable levels of pY even in v-Src-transformed cells (SI Fig. 9). However, when we analyzed DLC1 binding to premature termination mutants of the C-terminal tensin fragment that includes both domains, we unexpectedly found that a mutant Fig. 1. DLC1 and tensin form a complex in mammalian cells. (A) DLC1 was containing the tensin SH2 domain but lacking the PTB domain cotransfected with GFP or GFP-tensin into 293 cells, and specifically detected bound DLC1 with an efficiency that was only partially reduced DLC1 was coimmunoprecipitated in the anti-GFP immunopellet (Left). Recip- compared with the starting fragment that contained both do- rocally, GFP-tensin transfected into H1299 cells was specifically detected by coimmunoprecipitation of endogenous DLC1 with a DLC1 antibody (Right). mains (data not shown). We therefore inferred that DLC1 (B) Extracts from mel 1011 cells, which contain endogenous DLC1 and tensin, predominantly binds the SH2 domain, while the reduced binding were immunoprecipitated with control IgG or a tensin antibody. Coprecipi- suggested that DLC1 might also bind the PTB domain. To verify tated DLC1 was detected by a DLC1 antibody (Upper). The blot was reprobed these possibilities directly, we constructed plasmids encoding with the tensin antibody (Lower). isolated tensin-SH2 and tensin-PTB domains, fused to GST (Fig. 2B), and determined that both were bound FL DLC1 and DLC3 (Fig. 2A). The SH2 domain also bound endogenous DLC1 in several biological processes, including cell migration. Here we H1299 cells (Fig. 2C). Furthermore, the DLC1 sequences that demonstrate that this interaction is biologically significant and bind the tensin PTB domain are located N terminal to those show that two distinct regions of DLC1 bind two distinct domains required for binding the tensin SH2 domain because a DLC1 of tensin. fragment composed of amino acids 1–400 bound the PTB domain as efficiently as FL DLC1 but did not bind the SH2 Results domain (SI Fig.