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Deleted in liver cancer 1 (DLC1) utilizes a novel binding site for Tensin2 PTB domain interaction and is required for
Lo-Kong Chan1, Frankie Chi Fat Ko, Irene Oi-Lin Ng
1Department of Pathology, Liver Cancer and Hepatitis Research Laboratory and SH Ho Foundation Research Laboratories, The University of Hong Kong, Pokfulam, Hong Kong, China.
Background:
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. 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 Findings:
We 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 (DLC1DeltaPTB). 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/Significance:
This 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.
Insights
Deleted in liver cancer 1 (DLC1) interacts with tensin2 via a novel PTB domain binding site, impacting its RhoGAP activity and growth-suppressive functions in cancer. This clarifies DLC1-tensin interactions.
Area of Science:
- Cell Biology
- Molecular Oncology
- Protein Interactions
Background:
- Deleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently lost in hepatocellular carcinoma (HCC) and other cancers.
- DLC1 interacts with tensin proteins at focal adhesions, but the precise mechanisms remain unclear.
- Previous studies suggested a Src Homology 2 (SH2) domain-dependent interaction mechanism.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying DLC1 interactions with tensin family members.
- To identify novel binding sites and domains involved in DLC1-tensin interactions.
- To investigate the functional consequences of these interactions on DLC1 activity and cellular processes.
Main Methods:
- Co-immunoprecipitation assays were employed to identify protein-protein interactions.
- Site-directed mutagenesis was used to create DLC1 mutants lacking specific binding domains (DLC1DeltaPTB).
- Immunofluorescence microscopy was utilized to assess protein localization at focal adhesions.
Main Results:
- A novel binding site (375-385) on DLC1 was identified, primarily interacting with the phosphotyrosine binding (PTB) domain of tensin2.
- Disruption of this PTB binding site abolished DLC1-tensin2 interaction but did not affect interactions with tensin1 or cten.
- Loss of the PTB binding site partially reduced DLC1's RhoGAP activity and attenuated its growth-suppressive function.
Conclusions:
- DLC1 interacts with tensin2 in a PTB domain-dependent manner, distinct from previously described SH2-dependent interactions.
- This novel interaction site is crucial for maintaining DLC1's RhoGAP activity and its role in suppressing tumor cell growth.
- The findings provide new insights into the complex regulatory network of DLC1 in cancer progression.
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