C20, a novel binding partner of DLC1 identified by yeast-2-hybrid screening

Abstract

Hepatocellular carcinoma (HCC) is the fifth most common malignancies worldwide. Although the risk factors are known, the molecular and cellular mechanism of hepatocarcinogenesis is still unclear. Deleted in Liver Cancer 1(DLC1) is a recently identified tumor suppressor gene and the DLC1 gene encodes a RhoGAP protein, switching the active GTP bound form of small monomeric G-protein, Rho, into the inactive GDP bound form. We have previously shown that it is frequently underexpressed in HCC. We have also characterized DLC1 to be a growth suppressor in HCC cell lines and documented its in vitro RhoGAP activity. Our recent findings have also revealed that inhibition of Rho-mediated actin stress fiber formation by DLC1 is associated with its growth suppressive effect. Although DLC1 has multiple domains, the potential molecular targets and binding partners remain unclear. With the employment of yeast-2-hybrid system to screen against the human liver cDNA library, we have identified a yeast clone, namely clone 20 (C20), as a novel binding partner of DLC1. C20 contains a partial, unknown protein encoding DNA sequence of approximately 600 base pairs corresponding to human chromosome 19p13.3. C20 has a high sequence homology to the mouse Gtrgeo22 gene, which encodes a novel transmembrane protein containing dileucine and tyrosine-based motifs. Previous report has pointed out that mutation of Gtrgeo22can cause multiple defects in mice including male sterility. C20 is expressed in human liver and the transcripts are detectable by RT-PCR in all 10 HCC cell lines we have screened. The minimal interacting domain of DLC1 with C20 protein was mapped to the residues 293-647 of DLC1 in SFY526 yeast cells. Our results showed that DLC1 specifically interacted with C20, which is therefore a novel binding partner of DLC1. Further studies will derive new insight into the biological functions of DLC1. (This study was funded in part by the Michael Kadoorie Cancer Genetic Research Program of the Kadoorie Charitable Foundation)