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• W2128098893 abstract "•DEPDC1B is a cell-cycle gene involved in the transition from G2 phase to mitosis•Persistent adhesion at G2 phase delays CycB/CDK1 activation and G2/M transition•DEPDC1B controls RhoA/ROCK-dependent adhesion dynamics at G2 phase•DEPDC1B inhibits RhoA activation by displacing it from the PTPRF/GEF-H1 complex Cells entering mitosis become rounded, lose attachment to the substrate, and increase their cortical rigidity. Pivotal to these events is the dismantling of focal adhesions (FAs). How mitotic reshaping is linked to commitment to divide is unclear. Here, we show that DEPDC1B, a protein that accumulates in G2, coordinates de-adhesion events and cell-cycle progression at mitosis. DEPDC1B functions as an inhibitor of a RhoA-based signaling complex, which assembles on the FA-associated protein tyrosine phosphatase, receptor type, F (PTPRF) and mediates the integrity of FAs. By competing with RhoA for the interaction with PTPRF, DEPDC1B promotes the dismantling of FAs, which is necessary for the morphological changes preceding mitosis. The circuitry is relevant in whole organisms, as shown by the control exerted by the DEPDC1B/RhoA/PTPRF axis on mitotic dynamics during zebrafish development. Our results uncover an adhesion-dependent signaling mechanism that coordinates adhesion events with the control of cell-cycle progression. Cells entering mitosis become rounded, lose attachment to the substrate, and increase their cortical rigidity. Pivotal to these events is the dismantling of focal adhesions (FAs). How mitotic reshaping is linked to commitment to divide is unclear. Here, we show that DEPDC1B, a protein that accumulates in G2, coordinates de-adhesion events and cell-cycle progression at mitosis. DEPDC1B functions as an inhibitor of a RhoA-based signaling complex, which assembles on the FA-associated protein tyrosine phosphatase, receptor type, F (PTPRF) and mediates the integrity of FAs. By competing with RhoA for the interaction with PTPRF, DEPDC1B promotes the dismantling of FAs, which is necessary for the morphological changes preceding mitosis. The circuitry is relevant in whole organisms, as shown by the control exerted by the DEPDC1B/RhoA/PTPRF axis on mitotic dynamics during zebrafish development. Our results uncover an adhesion-dependent signaling mechanism that coordinates adhesion events with the control of cell-cycle progression. The cell cycle is a sequence of coordinated events leading to genome duplication and its correct segregation into the daughter cells at mitosis. The fidelity of this process is secured by mechanisms that are activated at specific restriction points: the cellular checkpoints (Gérard and Goldbeter, 2009Gérard C. Goldbeter A. Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle.Proc. Natl. Acad. Sci. USA. 2009; 106: 21643-21648Crossref PubMed Scopus (174) Google Scholar, Hartwell and Weinert, 1989Hartwell L.H. Weinert T.A. Checkpoints: controls that ensure the order of cell cycle events.Science. 1989; 246: 629-634Crossref PubMed Scopus (2418) Google Scholar, Tyson and Novak, 2008Tyson J.J. Novak B. Temporal organization of the cell cycle.Curr. Biol. 2008; 18: R759-R768Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The G2/M checkpoint occurs at the onset of mitosis and is in charge of preserving genomic integrity and its inheritance without damage or mutations (Branzei and Foiani, 2008Branzei D. Foiani M. Regulation of DNA repair throughout the cell cycle.Nat. Rev. Mol. Cell Biol. 2008; 9: 297-308Crossref PubMed Scopus (899) Google Scholar, Löbrich and Jeggo, 2007Löbrich M. Jeggo P.A. The impact of a negligent G2/M checkpoint on genomic instability and cancer induction.Nat. Rev. Cancer. 2007; 7: 861-869Crossref PubMed Scopus (455) Google Scholar). The G2/M transition is driven by several mitotic kinases, including the Aurora, Polo, and the cyclin-dependent kinases (CDKs) (Hochegger et al., 2008Hochegger H. Takeda S. Hunt T. Cyclin-dependent kinases and cell-cycle transitions: does one fit all?.Nat. Rev. Mol. Cell Biol. 2008; 9: 910-916Crossref PubMed Scopus (383) Google Scholar, Lindqvist et al., 2009Lindqvist A. Rodríguez-Bravo V. Medema R.H. The decision to enter mitosis: feedback and redundancy in the mitotic entry network.J. Cell Biol. 2009; 185: 193-202Crossref PubMed Scopus (414) Google Scholar, Smits and Medema, 2001Smits V.A. Medema R.H. Checking out the G(2)/M transition.Biochim. Biophys. Acta. 2001; 1519: 1-12Crossref PubMed Scopus (307) Google Scholar). The activation of the CDK1/cyclin B complex (mitosis-promoting factor [MPF]) is key in the control of mitotic entry and depends on multiple mechanisms that modulate the expression and/or localization of cyclin B and the phosphorylation status of CDK1 (Gavet and Pines, 2010Gavet O. Pines J. Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis.Dev. Cell. 2010; 18: 533-543Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, Lindqvist et al., 2009Lindqvist A. Rodríguez-Bravo V. Medema R.H. The decision to enter mitosis: feedback and redundancy in the mitotic entry network.J. Cell Biol. 2009; 185: 193-202Crossref PubMed Scopus (414) Google Scholar, Nigg, 2001Nigg E.A. Mitotic kinases as regulators of cell division and its checkpoints.Nat. Rev. Mol. Cell Biol. 2001; 2: 21-32Crossref PubMed Scopus (1249) Google Scholar, Norbury et al., 1991Norbury C. Blow J. Nurse P. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar, Santos et al., 2012Santos S.D. Wollman R. Meyer T. Ferrell Jr., J.E. Spatial positive feedback at the onset of mitosis.Cell. 2012; 149: 1500-1513Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Once activated, the MPF phosphorylates a series of molecular targets that trigger downstream mitotic events, such as nuclear envelope breakdown and chromosome condensation (Nigg, 2001Nigg E.A. Mitotic kinases as regulators of cell division and its checkpoints.Nat. Rev. Mol. Cell Biol. 2001; 2: 21-32Crossref PubMed Scopus (1249) Google Scholar, Ohi and Gould, 1999Ohi R. Gould K.L. Regulating the onset of mitosis.Curr. Opin. Cell Biol. 1999; 11: 267-273Crossref PubMed Scopus (165) Google Scholar). At mitotic entry, cells also become rounded, lose attachments to the substrate, and display increased cortical rigidity (Cramer and Mitchison, 1997Cramer L.P. Mitchison T.J. Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding.Mol. Biol. Cell. 1997; 8: 109-119Crossref PubMed Scopus (96) Google Scholar, Kunda and Baum, 2009Kunda P. Baum B. The actin cytoskeleton in spindle assembly and positioning.Trends Cell Biol. 2009; 19: 174-179Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, Théry and Bornens, 2006Théry M. Bornens M. Cell shape and cell division.Curr. Opin. Cell Biol. 2006; 18: 648-657Crossref PubMed Scopus (241) Google Scholar). This reshaping is thought to be necessary to set the axes for symmetric or asymmetric partitioning of cell determinants and to establish a correct spindle orientation (Kunda and Baum, 2009Kunda P. Baum B. The actin cytoskeleton in spindle assembly and positioning.Trends Cell Biol. 2009; 19: 174-179Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, Théry et al., 2005Théry M. Racine V. Pépin A. Piel M. Chen Y. Sibarita J.B. Bornens M. The extracellular matrix guides the orientation of the cell division axis.Nat. Cell Biol. 2005; 7: 947-953Crossref PubMed Scopus (637) Google Scholar). Adhesion to the extracellular matrix (ECM) is mainly mediated by structures called focal adhesions (FAs), in which establishment, maturation, and dismantling are tightly controlled (Parsons et al., 2010Parsons J.T. Horwitz A.R. Schwartz M.A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension.Nat. Rev. Mol. Cell Biol. 2010; 11: 633-643Crossref PubMed Scopus (1356) Google Scholar, Zamir and Geiger, 2001Zamir E. Geiger B. Molecular complexity and dynamics of cell-matrix adhesions.J. Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar). FAs exert a mechanostructural role by physically connecting the actin cytoskeleton to ECM via integrin receptors, and a signaling role, serving as hubs to assemble signaling complexes (Mitra and Schlaepfer, 2006Mitra S.K. Schlaepfer D.D. Integrin-regulated FAK-Src signaling in normal and cancer cells.Curr. Opin. Cell Biol. 2006; 18: 516-523Crossref PubMed Scopus (1181) Google Scholar, Parsons et al., 2010Parsons J.T. Horwitz A.R. Schwartz M.A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension.Nat. Rev. Mol. Cell Biol. 2010; 11: 633-643Crossref PubMed Scopus (1356) Google Scholar). As cells approach mitosis, they dismantle FAs via inactivation of FA kinase (FAK) and downmodulation of Rap1-GTPase activity (Dao et al., 2009Dao V.T. Dupuy A.G. Gavet O. Caron E. de Gunzburg J. Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis.J. Cell Sci. 2009; 122: 2996-3004Crossref PubMed Scopus (65) Google Scholar, Kunda and Baum, 2009Kunda P. Baum B. The actin cytoskeleton in spindle assembly and positioning.Trends Cell Biol. 2009; 19: 174-179Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, Pugacheva et al., 2006Pugacheva E.N. Roegiers F. Golemis E.A. Interdependence of cell attachment and cell cycle signaling.Curr. Opin. Cell Biol. 2006; 18: 507-515Crossref PubMed Scopus (44) Google Scholar, Yamakita et al., 1999Yamakita Y. Totsukawa G. Yamashiro S. Fry D. Zhang X. Hanks S.K. Matsumura F. Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis-specific serine phosphorylation of FAK.J. Cell Biol. 1999; 144: 315-324Crossref PubMed Scopus (106) Google Scholar). Concomitantly, cells experience mitotic rounding and cortical stiffening caused by actomyosin remodeling through RhoA (Maddox and Burridge, 2003Maddox A.S. Burridge K. RhoA is required for cortical retraction and rigidity during mitotic cell rounding.J. Cell Biol. 2003; 160: 255-265Crossref PubMed Scopus (228) Google Scholar, Matthews et al., 2012Matthews H.K. Delabre U. Rohn J.L. Guck J. Kunda P. Baum B. Changes in Ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression.Dev. Cell. 2012; 23: 371-383Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), ezrin, radixin, and moesin complex (ERM) proteins (Carreno et al., 2008Carreno S. Kouranti I. Glusman E.S. Fuller M.T. Echard A. Payre F. Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells.J. Cell Biol. 2008; 180: 739-746Crossref PubMed Scopus (182) Google Scholar), and myosin II (Maddox and Burridge, 2003Maddox A.S. Burridge K. RhoA is required for cortical retraction and rigidity during mitotic cell rounding.J. Cell Biol. 2003; 160: 255-265Crossref PubMed Scopus (228) Google Scholar). A mechanistic picture of how the cell coordinates detachment/rounding and entry into mitosis is, however, still lacking. Here we show that DEPDC1B, a cell-cycle-regulated gene (Nicassio et al., 2005Nicassio F. Bianchi F. Capra M. Vecchi M. Confalonieri S. Bianchi M. Pajalunga D. Crescenzi M. Bonapace I.M. Di Fiore P.P. A cancer-specific transcriptional signature in human neoplasia.J. Clin. Invest. 2005; 115: 3015-3025Crossref PubMed Scopus (16) Google Scholar), mediates the interplay between cell-cycle progression and de-adhesion events at the mitotic entry. The DEPDC1B protein specifically accumulates at the G2 phase of the cell cycle and inhibits RhoA recruitment to and activation by the FA-associated receptor protein tyrosine phosphatase, receptor type, F (PTPRF). By this mechanism, DEPDC1B functions as an inhibitor of the RhoA/Rho-associate protein kinase (ROCK)/MLC2 pathway during the G2/M transition, thereby allowing FA dismantling and cell detachment. Ablation of DEPDC1B impaired de-adhesion events and delayed mitotic entry. Similarly, conditions that induced persistent adhesion to the substrate, independently of DEPDC1B, inhibited mitotic entry, suggesting that adhesion per se controls cell-cycle progression. Thus, we have identified a feedback loop in which the nucleus signals to cell periphery the need to initiate mitotic reshaping through the synthesis of DEPDC1B. In turn, adhesion-dependent mechanisms delay progression into the M phase until mitotic reshaping is correctly executed. DEPDC1B is a proliferation-associated gene expressed in a cell-cycle-dependent fashion through an Rb/E2F-dependent transcriptional mechanism (Nicassio et al., 2005Nicassio F. Bianchi F. Capra M. Vecchi M. Confalonieri S. Bianchi M. Pajalunga D. Crescenzi M. Bonapace I.M. Di Fiore P.P. A cancer-specific transcriptional signature in human neoplasia.J. Clin. Invest. 2005; 115: 3015-3025Crossref PubMed Scopus (16) Google Scholar). We examined the pattern of expression of DEPDC1B mRNA and protein in HeLa cells synchronized by double-thymidine block (D-THY; Figure S1A available online). As cells entered the G2 phase (4 hr after release), DEPDC1B mRNA was induced, and the protein accumulated until mitosis (M phase, 8 hr), closely resembling the behavior of cyclin B. In addition, similar to cyclin B, DEPDC1B protein was degraded during mitosis in a proteasome-dependent manner (Hershko, 1999Hershko A. Mechanisms and regulation of the degradation of cyclin B.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354 (discussion 1575–1576): 1571-1575Crossref PubMed Scopus (81) Google Scholar) (Figure S1B). Knockdown (KD) of DEPDC1B with three different short interfering RNA (siRNA) oligos (1B-KD1, 1B-KD2, 1B-KD3; Figures 1A, 1B, and S1C) in HeLa cells synchronized by D-THY reduced the number of cells that reached mitosis (Figures 1A–1C; Movie S1), an effect that could be rescued by the concomitant expression of a siRNA resistant GFP-tagged DEPDC1B (Figures 1B and 1C). Flow-cytometry analysis showed that DEPDC1B-KD cells progressed normally from S to G2 (G2 phase, Figure 1D), while the transition from G2 to mitosis (mitosis, Figure 1D) was inhibited. Silencing of DEPDC1B also inhibited mitotic entry in other cell types, including nontransformed and cancer cell lines (Figure S1D). A DEPDC1B-like gene, DEPDC1A, encodes two isoforms (Figure S1E) whose expression is also regulated during the cell cycle (Figure S1F). Silencing of DEPDC1A caused a mitotic phenotype similar to that of DEPDC1B-KD (Figures 1C, S1G, and S1H). Importantly, the simultaneous depletion of both genes had additive and robust effects (Figures 1C, S1G, and S1H), arguing for functional redundancy and tight cooperative control over the G2/M transition. We investigated the effects of DEPDC1B silencing on the key molecular events of the G2/M transition (Güttinger et al., 2009Güttinger S. Laurell E. Kutay U. Orchestrating nuclear envelope disassembly and reassembly during mitosis.Nat. Rev. Mol. Cell Biol. 2009; 10: 178-191Crossref PubMed Scopus (341) Google Scholar, Lindqvist et al., 2009Lindqvist A. Rodríguez-Bravo V. Medema R.H. The decision to enter mitosis: feedback and redundancy in the mitotic entry network.J. Cell Biol. 2009; 185: 193-202Crossref PubMed Scopus (414) Google Scholar). Upon DEPDC1B silencing, the nuclear membrane abnormally persisted in the majority of cells (lamin B staining, Figure 1E), while no differences were found in cyclin B expression (Figures 1F–1G). However, nuclear accumulation of cyclin B was decreased (Figure 1F), suggesting that activation of the MPF could be impaired. Thus, we investigated the phosphorylation status of CDK1 since dephosphorylation on Tyr14/15 is required for progression into mitosis (Hunter, 1995Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling.Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2594) Google Scholar). The phosphorylation of CDK1 in DEPDC1B-KD cells was increased and sustained in time compared with control cells, confirming that MPF activation was delayed (Figure 1G). Together these results indicate that DEPDC1B is a regulator of the G2/M transition, acting upstream of the MPF activation. We employed the GFP-tagged version of DEPDC1B to analyze its subcellular distribution. In G2-syncronized cells, we observed a plasma membrane (PM) localization of DEPDC1B that persisted during mitosis (Figures S2A and S2B). In addition, while control cells lost attachment to the substrate and became rounded as they approached mitosis, DEPDC1B-KD cells appeared flattened, more motile, and often failed to detach from the substrate and become rounded (Movie S1). DEPDC1B might, therefore, act at the PM to regulate cellular adhesion. We investigated this possibility by following the dynamics of GFP-paxillin, a marker of FAs (Parsons et al., 2010Parsons J.T. Horwitz A.R. Schwartz M.A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension.Nat. Rev. Mol. Cell Biol. 2010; 11: 633-643Crossref PubMed Scopus (1356) Google Scholar, Zamir and Geiger, 2001Zamir E. Geiger B. Molecular complexity and dynamics of cell-matrix adhesions.J. Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar). In control cells, the typical punctuate staining of GFP-paxillin at the ventral membrane, which marks FAs in interphase, quickly disappeared as cells approached mitosis (Figure 2A; Movie S2). Conversely, DEPDC1B-KD cells displayed larger FAs that persisted in G2 (Figure 2A; Movie S2). A quantitative analysis revealed that the absolute number of FAs per cell was unaffected; however, their size was significantly increased and their mitotic dismantling delayed (Figures 2A, 2B, S2C, and S2D). We also detected significant modifications of actin dynamics in DEPDC1B-KD HeLa cells in G2 phase, with cells displaying an altered pattern of actin stress fibers and increased phosphorylation of the actin regulator myosin light chain 2 (MLC2-Ser19), a typical downstream target of the ROCK, and cofilin (pCofilin-Ser3) (Figures 2C and 2D). These observations could be extended to other cell types, including fibroblasts and nontransformed epithelial cells (Figure 2E). Finally, the silencing of DEPDC1B also altered the dynamics of cells spreading, an effect that could be rescued by the ectopic expression of the siRNA-resistant GFP-DEPDC1B (Figures 2F and S2E). These results point to a role for DEPDC1B in the control of cellular adhesion and actin dynamics during the G2 phase of the cell cycle. We investigated the relationship between the mitotic and the adhesion phenotypes caused by DEPDC1B silencing. Initially, we took advantage of a HeLa derivative clone (HeLa-S3) adapted to growth in suspension (Puck et al., 1956Puck T.T. Marcus P.I. Cieciura S.J. Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer.J. Exp. Med. 1956; 103: 273-283Crossref PubMed Scopus (519) Google Scholar) (Figure 3A). In these cells, DEPDC1B-KD did not affect the G2/M transition (Figure 3B), arguing that in the absence of cell adhesion the mitotic phenotype of DEPDC1B-KD cells could be suppressed. If so, it should be possible to abrogate the said phenotype by directly interfering with FAs. Thus, we silenced structural (vinculin, alpha-actinin) and catalytic (FAK) components of FAs in DEPDC1B-silenced cells (Figure 3C). In all cases, the downmodulation of FA components completely rescued the DEPDC1B-KD-dependent mitotic delay (Figure 3D), indicating that, in DEPDC1B-KD cells, the cell-cycle phenotype is linked to the abnormal persistence of FAs at the G2/M transition phase. We also analyzed the involvement of actomyosin contractility, which was altered upon DEPDC1B silencing (see Figure 2). As mentioned above, this process is closely linked to cell adhesion mechanisms and is controlled by RhoA/ROCK/MLC2 signaling. Therefore, we treated G2 synchronized DEPDC1B-KD cells with a ROCK inhibitor (Y27632, 10 μM). The treatment normalized the levels of phospho-MLC2 and concomitantly rescued the mitotic phenotype in DEPDC1B-KD cells (Figures 3E and 3F). If the impairment in G2/M transition observed in DEPDC1B-KD cells were indeed due to the persistence of FAs, then induction of persistent adhesive structures should phenocopy the DEPDC1B silencing. To investigate this, we employed two tools: (1) an autoinhibition-deficient mutant of vinculin (VincT12 mutant), which increases adhesion strength and force transmission (Humphries et al., 2007Humphries J.D. Wang P. Streuli C. Geiger B. Humphries M.J. Ballestrem C. Vinculin controls focal adhesion formation by direct interactions with talin and actin.J. Cell Biol. 2007; 179: 1043-1057Crossref PubMed Scopus (643) Google Scholar), and (2) manganese treatment (Mn2+, 1 mM), which induces ανβ3 integrin activation and clustering (Cluzel et al., 2005Cluzel C. Saltel F. Lussi J. Paulhe F. Imhof B.A. Wehrle-Haller B. The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells.J. Cell Biol. 2005; 171: 383-392Crossref PubMed Scopus (275) Google Scholar, Gailit and Ruoslahti, 1988Gailit J. Ruoslahti E. Regulation of the fibronectin receptor affinity by divalent cations.J. Biol. Chem. 1988; 263: 12927-12932Abstract Full Text PDF PubMed Google Scholar). In G2-synchronized HeLa cells, both treatments induced cell spreading on the substrate, formation of actin stress fibers, and high levels of phospho-MLC2 (Figures 3G and S3A–S3D), while concomitantly inhibiting the G2/M transition (Figures 3H–3I and S3E). These results suggest the existence of a DEPDC1B-based mechanism that controls the coordination of adhesion and actin cytoskeleton dynamics with entry into mitosis. Of note, the G2/M arrest induced by DNA damage-inducing agents (i.e., doxorubicin) appeared stronger than the cell-cycle arrest induced by persistent adhesion (Figures S3E and 3F). Since both actin cytoskeleton and adhesion dynamics are regulated by Rho-GTPases, we silenced the expression of each of the three prototypical members of this family, RhoA, Rac1, and Cdc42, alone and in conjunction with DEPDC1B-KD. The depletion of any of the three Rho-GTPases alone had no major effect on G2/M transition (Figure 4A). However, RhoA silencing, but not Rac1 or Cdc42 silencing, completely rescued the mitotic delay induced by DEPDC1B silencing (Figures 4A and 4B). Furthermore, the significant increase in the size of FAs in DEPDC1B-KD cells (see Figures 2A and 2B) was fully rescued by silencing RhoA (Figures 4C–4E). Finally, the DEPDC1B-KD-dependent spreading defect was rescued by silencing of RhoA, but not of Rac1 or Cdc42 (Figure S4A). These results link the function of DEPDC1B to the control of RhoA activity, likely through inhibition of the latter at the G2/M transition. This notion is further supported by the observations that (1) the DEPDC1B silencing increased the activity of RhoA (RBD pull-down assay, Figure 2D), (2) the levels of downstream targets, such as phospho-MLC2 (Figures 2C–2E), (3) RhoA overexpression in G2 cells phenocopied DEPDC1B KD, causing an increase in the size of FAs and in the levels of phospho-MLC2 (Figures 4F–4G) and a decrease in the mitotic index (Figure 4H). Despite having a RhoGAP-like (GTPase-activating) domain (Figure S1E), DEPDC1B is most likely not an active GAP since it lacks the catalytic arginine typical of true RhoGAPs (Graham et al., 1999Graham D.L. Eccleston J.F. Lowe P.N. The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride.Biochemistry. 1999; 38: 985-991Crossref PubMed Scopus (69) Google Scholar, Rittinger et al., 1997Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue.Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar) (Figure S4B). Indeed, we failed to detect RhoGAP activity of the recombinant RhoGAP domain of DEPDC1B (Figure S4C). To understand how DEPDC1B modulates RhoA signaling, we performed a yeast-two-hybrid (Y2H) screening to identify DEPDC1B-interacting proteins. Most of the hits were represented by PTPRF (Figure 5A; Table S1), a transmembrane receptor that has been suggested to function as a molecular hub at adhesive sites that coordinates adhesion and migration events (Chagnon et al., 2004Chagnon M.J. Uetani N. Tremblay M.L. Functional significance of the LAR receptor protein tyrosine phosphatase family in development and diseases.Biochem. Cell Biol. 2004; 82: 664-675Crossref PubMed Scopus (129) Google Scholar, Serra-Pagès et al., 1995Serra-Pagès C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions.EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (293) Google Scholar, Tsujikawa et al., 2002Tsujikawa K. Ichijo T. Moriyama K. Tadotsu N. Sakamoto K. Sakane N. Fukada S. Furukawa T. Saito H. Yamamoto H. Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule.Mol. Cancer Res. 2002; 1: 155-163PubMed Google Scholar). We confirmed the direct biochemical interaction between the DEPDC1B and PTPRF by glutathione S transferase (GST) pull-down experiments performed with cell lysates (Figures 5B and 5C) or purified proteins (Figure 5D). The interaction was also confirmed in intact cells by coimmunoprecipitation of GFP-tagged DEPDC1B and overexpressed PTPRF (Figure S5A) and by colocalization of the two proteins at the PM (Figure S5B). Next, we investigated whether PTPRF is involved in the DEPDC1B-dependent phenotypes. PTPRF-KD impaired cell spreading onto fibronectin, as previously shown (Figure S5C; Asperti et al., 2009Asperti C. Astro V. Totaro A. Paris S. de Curtis I. Liprin-alpha1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins.J. Cell Sci. 2009; 122: 3225-3232Crossref PubMed Scopus (49) Google Scholar), while it did not cause appreciable effects on the rate of mitotic entry (Figures 5E and 5F). However, PTPRF silencing was able to rescue both the mitotic delay and the increase in phospho-MLC2 levels induced by DEPDC1B silencing (Figures 5E and 5F), suggesting its participation in the DEPDC1B-dependent mechanisms that control actin dynamics at the G2/M transition. PTPRF phosphatase activity appeared not to be involved since PTPRF interaction with DEPDC1B was not affected by phosphatase treatment and treatment with inhibitors of PTPRF phosphatase activity did not significantly affect mitotic entry alone or together with DEPDC1B silencing (Figures S6A and S6B). To gain further insights into the role of PTPRF, we examined the PTPRF interactome by mass spectrometry. The list of PTPRF-interacting proteins was significantly enriched in components of the RhoA/ROCK pathway (p < 0.001; Figures 5G and S5D), including RhoA itself and several RhoA-binding partners, such as its effectors ROCK2 and mDIA1, as well as members of the actin network (Table S2). The direct interaction between PTPRF and RhoA was confirmed in in vitro pull-down experiments, largely independently of the activation status of the latter (Figures 5H and S5E) and by colocalization at the PM (Figure S6C). Importantly, the silencing of PTPRF reversed the mitotic defect induced by RhoA overexpression, albeit not completely (Figures 5I and 5J). Among PTPRF-interacting proteins, we identified several guanine nucleotide exchange factors (GEFs), and most notably GEF-H1, suggesting that the PTPRF/GEF-H1 axis could be involved in RhoA activation at FAs. This contention is reinforced by the observations that (1) PTPRF localizes at the PM close to FA markers (vinculin, paxillin) and to RhoA (Figures S6C–S6E); (2) the interaction between GEF-H1 and PTPRF was confirmed in a pull-down assay (Figure 6A); (3) silencing of GEF-H1, but not of another GEF, PDZ-GEF, which was pulled down by PTPRF, rescued the mitotic phenotype induced by DEPDC1B silencing (Figure 6B); (4) overexpression of GEF-H1 phenocopied the effect of RhoA activation or DEPDC1B silencing, inhibiting mitotic entry and inducing actin stress fibers (Figure 6C). The sum of our results strongly supports a model in which RhoA/ROCK signaling in G2 phase is induced by PTPRF/GEF-H1 and is inhibited by DEPDC1B. One mechanism through which this might occur is competition of the interaction of RhoA with PTPRF by DEPDC1B, with ensuing inhibition of the RhoA signaling complex and the consequent dismantling of adhesion structures at the G2/M transition. We tested this hypothesis through a series of experiments. We showed by in vitro pull-down assays performed on total cellular lysates that the interaction between the cytoplasmic domain of PTPRF (GST-PTPRF-c) and RhoA was almost completely inhibited by the simultaneous presence of excess DEPDC1B (obtained by ectopic expression) in the cell lysate (Figure 6D). This effect on PTPRF and RhoA binding was also reproduced in stable isotope labeling by amino acids in cell culture (SILAC) experiments (Figure 6E) and appears specific for RhoA itself since binding of GEF-H1 (RhoA activator), ROCK2, and mDIA1 (RhoA effectors) to PTPRF were unaffected by DEPDC1B overexpression (Figures 6A and 6F). Conversely, silencing DEPDC1B significantly increased the interaction between GST-PTPRF-c and RhoA in the same assay (Figure 6G) and the interaction between RhoA and ROCK2 in the RBD pull-down assay (Figure 6H). Finally, the purified DEPDC1B fragment that interacts with PTPRF-c (Figure 5D) halved the interaction of the latter with RhoA in in vitro pull-down experiments (Figure 6I). These results suggest a role for the DE" @default.
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• W2128098893 date "2014-11-01" @default.
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• W2128098893 title "DEPDC1B Coordinates De-adhesion Events and Cell-Cycle Progression at Mitosis" @default.
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