DEPDC-1B MODULATES RHOA-DEPENDENT CELL ADHESION DURING MITOTIC ENTRY

One of the most frequent alterations occurring in cancer is the inactivation of the mechanisms controlling cell cycle progression: the cellular checkpoints. Among the different existing checkpoints, the one controlling the G2/M transition holds great interest because of its implications for genome stability and tumorigenesis. Previous work in our lab identified DEPDC-1B as an E1A-induced gene during cell-cycle re-entry of differentiated cells (Nicassio et al. 2005). In this study, DEPDC-1B was found a cell cycle regulated-gene, with transcriptional expression peaking at the G2 and M phases (Nicassio et al. 2005). The DEPDC-1B protein possesses the structural features of a signaling protein, with two conserved domains (a DEP and a Rho-GAP domain) putatively involved in RhoGTPase signaling. Therefore, we hypothesized the existence of a previously un-described signaling pathway involving both DEPDC-1B and Rho-GTPases in the control of G2/M transition. Our results identify DEPDC-1B as a key player in the regulation of mitotic entry in vertebrates. DEPDC-1B ablation causes a delay in mitotic entry in human cell lines and impairs mitotic progression in vivo during the early stages of Zebrafish development. By the use of biochemical and genetic approaches we provide evidence for a DEPDC- 1B/RhoA complex, possibly with the participation of the focal adhesion-specific receptor phosphatase PTPRF, able to regulate focal adhesion and cytoskeleton dynamics at the G2/M transition. Our model suggests that DEPDC-1B acts as a negative modulator of RhoA activity, and requires both the DEP and Rho-GAP domains, although the latter appears to be an atypical Rho-GAP that does not possess any GAP activity per se. We observed that, even in Zebrafish development, DEPDC-1B control on cell cycle progression is RhoA-dependent and involves both domains of DEPDC1B. Our results also indicate that alterations of adhesion during the G2 phase occur as a consequence of either DEPDC-1B/PTPRF silencing or RhoA hyper-activation. Such modulation of expression of the three genes is able to induce a delay in the G2/M transition. We, therefore, suggest the existence of an “Adhesion Checkpoint”, centered on the DEPDC-1B/RhoA axis, which controls cell detachment at the G2/M transition by the coordinated disassembly of focal adhesive structures. This previously uncharacterized mechanism appears to be essential for the proper progression to mitosis in vitro and in vivo and might potentially impact also on the mechanisms controlling DNA damage response at the G2/M phase and cellular transformation.