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. 2009 Jul 24;284(30):20061-9.
doi: 10.1074/jbc.M109.000885. Epub 2009 May 19.

Ubiquitin-mediated degradation of the formin mDia2 upon completion of cell division

Aaron D DeWard  1 , Arthur S Alberts
Affiliations

Ubiquitin-mediated degradation of the formin mDia2 upon completion of cell division

Aaron D DeWard et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
mDia2 localization and expression is cell cycle-dependent. A, HeLa cells were stained with rabbit anti-mDia2 (fluorescein isothiocyanate (FITC)) and phalloidin (Texas Red) or mDia2 (fluorescein isothiocyanate) and tubulin (TRITC) along with DNA (Hoechst, blue). Overlay shows the merged image of all three channels. Arrows in the top panels denote the actin-rich cleavage furrow containing mDia2. B, HeLa cell lysates were collected at time points indicated after double thymidine G1/S arrest and release. A represents lysate from asynchronous population of cells. Immunoblots (IB) were probed with anti-mDia2 (1358) and anti-EF1α as a loading control. C, cells from B were labeled as described under “Experimental Procedures” to determine DNA content and analyzed on a flow cytometer. Plots show cell numbers relative to DNA content. The percentages of cells in G1, S, or M phase are shown for each time point.
FIGURE 2.
FIGURE 2.
Proteasome inhibition prevents mDia2 degradation. A, HeLa cells were arrested with a double thymidine block and released into growth media. MG132 proteasome inhibitor (20 μm) was added to a population of cells upon thymidine release and incubated for 10 h (lane 6). Lysates were collected at the time points indicated and immunoblotted for mDia2. EF1α was probed as a loading control. A represents cell lysate from an asynchronous population. B, cells from A were labeled as described under “Experimental Procedures” to determine DNA content and analyzed on a flow cytometer. Plots show cell numbers relative to DNA content. The percentages of cells in G1, S, or M phase are shown for each time point. C, HeLa cells were thymidine arrested as in A (t = 0 h) but released into growth media containing 100 ng/ml nocodazole for 6 h (t = 0–6 h). Cells were then rinsed and released into normal growth media. MG132 (20 μm) was added to a population of cells 1 h after the nocodazole release and incubated for 5 h (lane 10). Immunoblotting of lysates were performed as in A. D, flow cytometry profiles showing DNA content of cells examined in C.
FIGURE 3.
FIGURE 3.
mDia2 is polyubiquitinated. A, HEK293T cells were transfected with Myc-mDia2, HA-ubiquitin, or co-transfected with both plasmids. Lysates were immunoprecipitated (IP) for either Myc or HA. Immunoblots (IB) of lysates and immunoprecipitations were probed with anti-Myc or anti-HA to examine the extent of ubiquitination. B, HeLa cells were incubated with 10 μm MG132 for 18 h. Lysates were immunoprecipitated using anti-β-catenin or anti-mDia2. Immunoblots were probed with anti-ubiquitin, anti-β-catenin, or anti-mDia2 (1358) to examine endogenous ubiquitination. C, HEK293T cells were co-transfected with Myc-mDia2 and HA-ubiquitin. Lysates were immunoprecipitated with anti-Myc. HeLa cells were incubated with 20 μm MG132 for 4 h. Lysates were immunoprecipitated with anti-β-catenin. Immunoprecipitations were immunoblotted with anti-Myc and an antibody specific to polyubiquitin chains.
FIGURE 4.
FIGURE 4.
mDia2 ubiquitination increases at the end of mitosis. A, HEK293T cells were co-transfected with Myc-mDia2 and HA-ubiquitin. Cells were arrested at G1/S phase with a double thymidine block. Cells were released into growth media, and lysates were collected at time points indicated and subjected to Myc immunoprecipitation (IP). Immunoblots (IB) were probed with Myc and HA to examine the extent of mDia2 ubiquitination. EF1α was blotted as a loading control, and cyclin E was blotted to verify progression through the cell cycle. B, cells from A were labeled as described under “Experimental Procedures” to determine DNA content and analyzed on a flow cytometer. Plots show cell numbers relative to DNA content. The percentages of cells in G1, S, or M phase are shown for each time point. C, HEK293T cells were co-transfected with Myc-mDia2 and HA-ubiquitin (second through seventh lanes) in addition to Myc-GFP and HA-ubiquitin as a negative control (first lane). Cells were incubated with 100 ng/ml nocodazole for 24 h to arrest cells in mitosis. Cells were rinsed, and lysates were collected at the indicated times after nocodazole washout. Immunoblots were probed with anti-Myc and anti-HA. D, flow cytometry profiles showing DNA content of cells from C.
FIGURE 5.
FIGURE 5.
Ubiquitin is attached to multiple residues on mDia2. A, ClustalW protein sequence alignment of mouse mDia1, human mDia1, mouse mDia2, and human mDia2 (accession numbers O08808, NP_005210, NP_062644, NP_001035982, respectively). Bold lysines represent ubiquitinated residues identified in Denis et al. (29). B, schematic profile of mDia2 mutants examined for ubiquitination. WT, wild type. C, lysine-to-arginine mutant versions of Myc-mDia2 were co-transfected with HA-ubiquitin in HEK293T cells. Lysates were Myc-immunoprecipitated (IP), and immunoblots (IB) were probed with anti-Myc and anti-HA. D, mDia2 mutants were co-transfected with HA-ubiquitin in HEK293T cells. Lysates were immunoprecipitated, and immunoblots were probed with anti-GFP, anti-Myc, or anti-HA to examine the extent of ubiquitination.
FIGURE 6.
FIGURE 6.
Deregulated mDia2 results in cytokinesis failure. A, bar graph showing the percent of multinucleate cells after injection with plasmids that encode EGFP-mDia2-ΔGBD/ΔDAD (amino acids 521–1040), EGFP-mDia2-ΔGBD/ΔDAD-I704A, EGFP-mDia2-DAD, or EGFP-mDia2-DAD-M1041A. Error bars represent the S.D. from three independent experiments (p < 0.01 (**) and p < 0.05 (*) using Student's t test for significance, n = 30). B, HeLa cells were microinjected with plasmid DNA encoding EGFP-mDia2-DAD, which interferes with mDia autoregulation. Cells were stained for F-actin (Texas Red) and nuclei (Hoechst, blue). C, HeLa cells were microinjected with a mutant version of the plasmid described in B that is unable to bind to DID and interfere with autoregulation (DAD-M1041A). Cells were stained as in B.
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References

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