Abstract
BackgroundDeleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently deleted and underexpressed in hepatocellular carcinoma (HCC) as well as in other cancers. Recent independent studies have shown interaction of DLC1 with members of the tensin focal adhesion protein family in a Src Homology 2 (SH2) domain-dependent mechanism. DLC1 and tensins interact and co-localize to punctate structures at focal adhesions. However, the mechanisms underlying the interaction between DLC1 and various tensins remain controversial.Methodology/Principal FindingsWe used a co-immunoprecipitation assay to identify a previously undocumented binding site at 375–385 of DLC1 that predominantly interacted with the phosphotyrosine binding (PTB) domain of tensin2. DLC1-tensin2 interaction is completely abolished in a DLC1 mutant lacking this novel PTB binding site (DLC1ΔPTB). However, as demonstrated by immunofluorescence and co-immunoprecipitation, neither the focal adhesion localization nor the interaction with tensin1 and C-terminal tensin-like (cten) were affected. Interestingly, the functional significance of this novel site was exhibited by the partial reduction of the RhoGAP activity, which, in turn, attenuated the growth-suppressive activity of DLC1 upon its removal from DLC1.Conclusions/SignificanceThis study has provided new evidence that DLC1 also interacts with tensin2 in a PTB domain-dependent manner. In addition to properly localizing focal adhesions and preserving RhoGAP activity, DLC1 interaction with tensin2 through this novel focal adhesion binding site contributes to the growth-suppressive activity of DLC1.
Highlights
The small, monomeric G-protein Rho has been classically defined as a key biological regulator of the actin cytoskeleton [1,2,3]
We demonstrated that the C-terminus tensin2 fragment, including the Src Homology 2 (SH2) and phosphotyrosine binding (PTB) domains (SH2-PTB in Fig. 1B), was sufficient to bind Deleted in liver cancer 1 (DLC1) (Fig. 1A)
In contrast to other reports, we found that removing the SH2 domain in tensin2 only partially reduced DLC1 binding
Summary
The small, monomeric G-protein Rho has been classically defined as a key biological regulator of the actin cytoskeleton [1,2,3]. Extensive studies have shown that DLC1 utilizes this RhoGAP activity to suppress cell proliferation [15,18,23,25,26,27,28,29], trigger apoptosis [25] and to reduce cell migration [26,28], cell invasion and the resultant cancer metastasis in cell lines as well as mouse models with different tissue origins [29,30,31]. Deleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently deleted and underexpressed in hepatocellular carcinoma (HCC) as well as in other cancers. Recent independent studies have shown interaction of DLC1 with members of the tensin focal adhesion protein family in a Src Homology 2 (SH2) domain-dependent mechanism. The mechanisms underlying the interaction between DLC1 and various tensins remain controversial
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