Ubiquitin-mediated Degradation of the Formin mDia2 upon Completion of Cell Division

Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTP-binding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component.Formin proteins play a role in diverse processes such as cell migration (1, 2), vesicle trafficking (3, 4), tumor suppression (5, 6), and microtubule stabilization (7, 8). Formins also play an essential and conserved role in cytokinesis (9–11). Proper cell division is essential in all animals to maintain the integrity of their genome. Failure to complete cytokinesis can result in genomic instability and ultimately lead to disease such as cancer (12).The members of the mDia² family of formins are autoregulated Rho effectors that remodel the cytoskeleton by nucleating and elongating non-branched actin filaments (13). The amino terminus of mDia contains a GTPase binding domain (GBD) that directs interaction with specific Rho small GTP-binding proteins. The adjacent Dia inhibitory domain (DID) mediates mDia autoregulation through its interaction with the carboxyl-terminal diaphanous autoregulatory domain (DAD) (14, 15). Between the DID and DAD domains lie the conserved formin homology 1 (FH1) and FH2 domains. The FH1 domain is a proline-rich region that mediates binding to other proteins such as profilin, Src, and Dia-interacting protein (16–19). In contrast, the FH2 domain binds monomeric actin to generate filamentous actin (F-actin) and can also bind microtubules directly to induce their stabilization (8, 20).Although the mechanism of mDia activation is well characterized, little is known about its inactivation. Previous reports have suggested that formins can cycle between active, partially active, and inactive states (21, 22) due to GTP hydrolysis upon Rho binding to GTPase-activating proteins. Another formin inactivation mechanism is through mDia interactions with Dia-interacting protein (23). In the context of cortical actin assembly, Dia-interacting protein negatively regulates mDia2 actin polymerization but has no effect on mDia1 actin polymerization despite its ability to interact with both proteins directly (17). Because of the fundamental role for formins in cell division, we sought to identify how mDia2 is inactivated in mitosis.During cell division, the expression level and activity of many proteins (e.g. cyclins and Aurora and Polo kinases) are tightly regulated (24). A unifying regulatory mechanism among these proteins is ubiquitin-mediated proteolysis. In this study we find that mDia2 protein levels are constant from S phase into mitosis and dramatically decrease at the end of mitosis due to ubiquitin-mediated degradation. Failure to inhibit mDia2 actin assembly results in multinucleation, which supports an essential role for the tight regulation of mDia2 during cell division.