Cyclin A/cdk2 Regulates Adenomatous Polyposis Coli-dependent Mitotic Spindle Anchoring
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.042820
ISSN1083-351X
AutoresHeather Beamish, Leonore de Boer, Nichole Giles, Frankie Stevens, Vanessa Oakes, Brian Gabrielli,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoMutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-β-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis. Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-β-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis. Adenomatous polyposis coli (APC) 5The abbreviations used are: APCadenomatous polyposis coliFACSfluorescent-activated cell sortingPBSphosphate-buffered salinePipes1,4-piperazinediethanesulfonic acidGSTglutathione S-transferaseWtwild type. was initially identified as a tumor suppressor in familial colon cancers. It is a regulator of Wnt-β-catenin signaling and thereby regulates progression into the cell cycle, but also has Wnt-independent mitotic roles in spindle anchoring and kinetochore function (1Aoki K. Taketo M.M. J. Cell Sci. 2007; 120: 3327-3335Crossref PubMed Scopus (365) Google Scholar, 2Näthke I. Nat. Rev. Cancer. 2006; 6: 967-974Crossref PubMed Scopus (115) Google Scholar, 3Rusan N.M. Peifer M. J. Cell Biol. 2008; 181: 719-726Crossref PubMed Scopus (45) Google Scholar). These latter functions of APC are mediated through its ability to bind microtubules and the end-binding protein, EB1 (2Näthke I. Nat. Rev. Cancer. 2006; 6: 967-974Crossref PubMed Scopus (115) Google Scholar). Loss or mutation of APC has been demonstrated to increase chromosomal instability, although whether this is through its Wnt-dependent or independent functions is unclear (3Rusan N.M. Peifer M. J. Cell Biol. 2008; 181: 719-726Crossref PubMed Scopus (45) Google Scholar). The mitotic defects caused by APC mutation and depletion are characterized by an inability to locate the center of the cell and failure of chromosomal alignment (4Green R.A. Wollman R. Kaplan K.B. Mol. Biol. Cell. 2005; 16: 4609-4622Crossref PubMed Scopus (188) Google Scholar). It was also associated with a loss of normal spindle orientation in small intestinal crypts of APCMin/+ mice (5Caldwell C.M. Green R.A. Kaplan K.B. J. Cell Biol. 2007; 178: 1109-1120Crossref PubMed Scopus (128) Google Scholar), suggesting that disruption of the normal mitotic functions of APC are likely to be major contributors to the chromosomal instability observed. adenomatous polyposis coli fluorescent-activated cell sorting phosphate-buffered saline 1,4-piperazinediethanesulfonic acid glutathione S-transferase wild type. APC interaction with EB1 is regulated by phosphorylation of its C-terminal domain by cyclin B/cdk1 in mitosis (6Nakamura M. Zhou X.Z. Lu K.P. Curr. Biol. 2001; 11: 1062-1067Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 7Askham J.M. Moncur P. Markham A.F. Morrison E.E. Oncogene. 2000; 19: 1950-1958Crossref PubMed Scopus (112) Google Scholar). The majority of APC mutations occurs in a region from codons 1,000 to 1,500 called the mutation cluster region (MCR) and result in truncations of the C-terminal half of the protein, which includes the β-catenin, microtubule, and EB1 binding sites of APC (1Aoki K. Taketo M.M. J. Cell Sci. 2007; 120: 3327-3335Crossref PubMed Scopus (365) Google Scholar, 2Näthke I. Nat. Rev. Cancer. 2006; 6: 967-974Crossref PubMed Scopus (115) Google Scholar). Depletion of either APC or EB1 produce almost identical mitotic defects, indicating their interaction is critical to normal spindle formation (4Green R.A. Wollman R. Kaplan K.B. Mol. Biol. Cell. 2005; 16: 4609-4622Crossref PubMed Scopus (188) Google Scholar, 8Draviam V.M. Shapiro I. Aldridge B. Sorger P.K. EMBO J. 2006; 25: 2814-2827Crossref PubMed Scopus (129) Google Scholar). However, expression of various truncation mutants across the MCR revealed interesting differences the spindle defects observed, suggesting that this role of APC in spindle function is not solely due to interaction with EB1 (4Green R.A. Wollman R. Kaplan K.B. Mol. Biol. Cell. 2005; 16: 4609-4622Crossref PubMed Scopus (188) Google Scholar). Progression into mitosis is regulated by cyclin B/cdk1, but the timing of its activation is regulated by cyclin A/cdk2 (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 10Fung T.K. Ma H.T. Poon R.Y. Mol. Biol. Cell. 2007; 18: 1861-1873Crossref PubMed Scopus (84) Google Scholar, 11Gong D. Pomerening J.R. Myers J.W. Gustavsson C. Jones J.T. Hahn A.T. Meyer T. Ferrell Jr., J.E. Curr. Biol. 2007; 17: 85-91Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 12Mitra J. Enders G.H. Oncogene. 2004; 23: 3361-3367Crossref PubMed Scopus (70) Google Scholar), which in turn is regulated by the dual specificity phosphatase cdc25B in G2 phase (13Goldstone S. Pavey S. Forrest A. Sinnamon J. Gabrielli B. Oncogene. 2001; 20: 921-932Crossref PubMed Scopus (76) Google Scholar). Cyclin A is destroyed at prometaphase (14den Elzen N. Pines J. J. Cell Biol. 2001; 153: 121-136Crossref PubMed Scopus (310) Google Scholar) suggesting that its activity is required for not only entry into mitosis but during the early part of mitosis itself. The majority of substrates identified for cyclin A/cdk2 are nuclear, where the majority of cyclin A/cdk2 is localized in G2, but reports also suggest that cyclin A is capable of localizing to both the cytoplasm and centrosomes (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 14den Elzen N. Pines J. J. Cell Biol. 2001; 153: 121-136Crossref PubMed Scopus (310) Google Scholar, 15Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (529) Google Scholar), thus there are likely to be additional substrates for this complex in the cytoplasmic compartment. In vitro studies using Xenopus extracts have demonstrated that cyclin A/cdk is capable of increasing microtubule nucleation at the centrosomes (16Buendia B. Draetta G. Karsenti E. J. Cell Biol. 1992; 116: 1431-1442Crossref PubMed Scopus (118) Google Scholar). Thus it is likely that cyclin A in association with its cdk partner has roles in not only promoting entry into mitosis but also in establishing mitosis, possibly by influencing the mitotic machinery. We have used siRNA to knockdown cyclin A2, the major cyclin A isoform in somatic cells, and cdk2 inhibitors to examine the role of the G2 phase cyclin A/cdk2 complex in cell cycle progression. We demonstrate that knockdown of cyclin A delayed progression through mitosis and activation of the spindle assembly checkpoint. Spindle anchoring was also defective, a phenotype identical to APC-truncating mutants. We demonstrate that cyclin A/cdk2 binds to APC in late G2 phase/early mitosis and phosphorylates Ser-1360, and that the lack of this phosphorylation of APC results in identical mitotic defects to the absence of cyclin A/cdk2. The cell lines, human cervical cancer cell line HeLa, HeLa cells stably expressing GFP-H2B or Cherry-tubulin as described previously (17Stevens F.E. Beamish H. Warrener R. Gabrielli B. Oncogene. 2008; 27: 1345-1354Crossref PubMed Scopus (73) Google Scholar), U2OS osteosarcoma cells, and primary neonatal foreskin fibroblasts (NFF) were cultured as described previously (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar). Cells were synchronized with a single thymidine block as described previously (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 18Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). Cells were collected for FACS analysis of DNA content as previously described (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 18Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). For immunoblotting, cells were lysed analyzed as previously described (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 17Stevens F.E. Beamish H. Warrener R. Gabrielli B. Oncogene. 2008; 27: 1345-1354Crossref PubMed Scopus (73) Google Scholar). Proteins were probed with the indicated antibodies and detected by enhanced chemiluminescent detection. For siRNA-mediated ablation of cyclin A, cells were transfected with three siRNAs using a scrambled control as described previously (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar). Cdc25B siRNA was purchased from Dharmacon. To generate a cyclin A expression clone, which was resistant to A1 siRNA, site-directed mutagenesis was carried out on pCherry-Cyclin A as per manufacturer's instructions (Stratagene). HeLa cells were transfected with the pCherry-resistant cyclin A construct as previously described. siRNA knockdown of cyclin A was carried out at 24-h post-transfection. Cells grown on poly-l-lysine-coated coverslips were treated as required and fixed in methanol at −20 °C. The cells were treated with 0.1% saponin/2% bovine serum albumin/PBS for 1 h at room temperature, before probing with the primary antibodies indicated in the figure legends in 2% bovine serum albumin/PBS at room temperature for 60 min; after washing in PBS, the cells were incubated with the appropriate secondary antibody conjugated with either fluorescein isothiocyanate (FITC) or Cy3. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) at 1 μg/ml. Confocal images were obtained on the Zeiss LSM 510 Meta confocal or the Zeiss Apoptome microscope (Zeiss, Goettingen, Germany) as indicated. Epi-fluorescence microscopy was carried out using a Zeiss Axioskop 2 plus microscope and a Zeiss Axiocam HRm camera. The following antibodies used in this study were purchased from the indicated commercial sources: rabbit polyclonal cyclin A and cdc25B (Santa Cruz Biotechnology), α-tubulin, and Aurora B and EB1 (BD Biosciences), MAD2 (Covance), and APC (Abcam). Polyclonal cyclin B1, cdk2, and cdk1 antibodies were made in-house as previously reported (13Goldstone S. Pavey S. Forrest A. Sinnamon J. Gabrielli B. Oncogene. 2001; 20: 921-932Crossref PubMed Scopus (76) Google Scholar, 18Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). To examine astral microtubules (MT), cells were fixed as follows: cells were first washed with PBS followed by MT stabilization buffer (80 mm Pipes pH 6.8, 3 mm EGTA, 1 mm MgCl2 in PBS) for ∼3 s at room temperature. This was replaced with 3% paraformaldehyde into which 1% glutaraldehyde was added after ∼3 s and incubated for 10 min at 37 °C. Cells were then incubated twice with 1 mg/ml NaBH4 in PBS for 2–7 min at 37 °C, washed with PBS, and stored at 4 °C in PBS. Time lapse experiments were performed using HeLa cells, and HeLa cells stably expressing GFP-H2B or Cherry-tubulin as previously described (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 17Stevens F.E. Beamish H. Warrener R. Gabrielli B. Oncogene. 2008; 27: 1345-1354Crossref PubMed Scopus (73) Google Scholar). To maintain cells in mitosis for an extended period of time, a non-destructible form of cyclin B, TAT-ΔN86 cyclin B, was added to the cells as previously described (19Warrener R. Beamish H. Burgess A. Waterhouse N.J. Giles N. Fairlie D. Gabrielli B. Faseb J. 2003; 17: 1550-1552Crossref PubMed Google Scholar). The APC fragment 1089–1370 was produced by PCR from full-length APC (pEF-myc-APC, gift from M. Faux, Ludwig Institute, Australia) and subcloned into pGEX-2T. Serine 1100 and/or serine 1360 were mutated to alanines by PCR in the pGEX fusion constructs and confirmed by sequencing. GST fusion products were expressed and purified as previously described (18Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). To produce full-length APC S 1360A mutant, the mutant fragment of GEX-APC-(1011–1470) was PCR-amplified then subcloned back into full-length APC (pEF-myc-APC) using unique restriction sites. To evaluate cyclin A-specific kinase activity and GST-APC-(1011–1470)-associated kinase, cells were lysed for 30 min at 4 °C with agitation in lysis buffer with 0.25 m NaCl. Equal amounts of protein (300–500 μg per sample) were immunoprecipitated with either anti-cyclin A and 30 μl of protein A-Sepharose or GST-APC bound to glutathione-Sepharose overnight at 4 °C. The precipitates were washed three times with NETN buffer and once with KB buffer (20 mm Tris, pH 7.5, 1 mm dithiothreitol) before incubating in 30 μl of RC buffer (20 mm Tris, pH 7.5, 15 mm MgCl2, 0.5 mg/ml histone H1, 5 μCi [γ-32P]ATP per sample) for 15 min at 30 °C. The labeled proteins were resolved on 10% SDS-PAGE and quantitated by phosphorimaging. The cdk inhibitor, roscovitine and the cdk2 inhibitor RO-03-9099 were used at a concentration of 0.5 μm. HeLa cells were transfected with siRNA A1 as previously described, 48-h post-transfection, the medium was gently removed and replated into fresh dishes to determine the number of viable cells present in the medium. The parental plate was harvested to determine levels of cyclin A by immunoblotting. Colonies were allowed to grow for 10 days, fixed in methanol/acetic acid (3:1) stained with crystal violet (0.4 mg/ml), and examined. We have developed three independent cyclin A-specific siRNAs that reduced cyclin A protein levels (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar) (Fig. 1A). This depletion was evident for up to 72 h after siRNA transfection (supplemental Fig. S1). The transfection efficiency was determined to be >90% when FITC-conjugated siRNA was used. Cyclin A depletion in asynchronous cultures increased the proportion of mitotic cells 24-h post-transfection, as determined by FACS analysis of MPM2 antibody staining of mitotic cells; 3.6% of cyclin A-depleted cells were in mitosis compared with 1.6% in controls. To further examine the effects of cyclin A knockdown on mitotic progression, cyclin A siRNA-transfected HeLa cells were synchronized in early S phase by thymidine block-and-release, and progress through the cell cycle was followed by time lapse microscopy. Depletion of cyclin A with any of the three cyclin A siRNAs consistently resulted in an extension in the duration of mitosis (Fig. 1B). HeLa cells transfected with the nonsense siRNA (Con) had a mean duration of 44 min in mitosis compared with 142 min in cyclin A knockdown cells (results for A1 siRNA knockdown). A proportion of cyclin A siRNA-treated cells had relatively normal length mitoses, which are likely to be cells where depletion failed. To demonstrate that this mitotic delay was specifically due to cyclin A depletion, cyclin A fused with Cherry fluorescent protein (Wt Cyclin A) was mutated to generate a clone resistant to the A1 siRNA (Res Cyclin A, Fig. 1C). Transfection of Wt Cyclin A into HeLa cells had little effect on transit through mitosis, while cyclin A knockdown using A1 siRNA in Wt Cyclin A-expressing cells increased the length of mitosis to an average of 227 min (Fig. 1D, p < 0.0001), similar to the effect of cyclin A depletion in parental HeLa cells (Fig. 1B). Expression of siRNA resistant Res Cyclin A in HeLa cells again had little effect on mitosis, while knockdown of the endogenous cyclin A and not the Res Cyclin A by siRNA A1 significantly rescued the mitotic delay normally observed (Fig. 1D, p < 0.0001). Together, the ability of three separate siRNA to induce a mitotic delay which was rescued by re-introduction of an siRNA-resistant form of cyclin A provides convincing evidence that the mitotic delay was consequence of the lack of cyclin A. Examination of the mitotic cells in cyclin A siRNA-treated cultures by immunofluorescence analysis of DNA and microtubules revealed normal bipolar spindles but a failure of some chromosomes to congress to the metaphase plate (Fig. 2A). Quantitation of proportion of cells with formed bipolar spindles and lagging chromosomes revealed an increase from 38 to 67% (p < 0.01; Fig. 2B). Using MAD2 staining as a marker of spindle assembly checkpoint activity (20Skoufias D.A. Andreassen P.R. Lacroix F.B. Wilson L. Margolis R.L. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4492-4497Crossref PubMed Scopus (239) Google Scholar) revealed that chromosomes that had failed to congress were stained for MAD2, indicating activation of the spindle checkpoint (Fig. 2C, lower panel), while cells without lagging chromosomes had no MAD2 staining (Fig. 2C, upper panel). This indicated activation of the spindle assembly checkpoint in response to retarded chromosome congression. Examination of other regulators of chromosome congression, the chromosomal passengers Aurora B, INCENP, survivin, and Borealin (21Vagnarelli P. Earnshaw W.C. Chromosoma. 2004; 113: 211-222Crossref PubMed Scopus (284) Google Scholar) revealed their normal accumulation at the centromeres was unaffected by cyclin A depletion (data not shown). Examination of cells at extended times after cyclin A knockdown revealed no increase in cells with multiple or fractured nuclei, indicators of spindle checkpoint failure. Time lapse microscopy of cyclin A knockdown in HeLa cells expressing GFP-H2B confirmed that cells delayed in mitosis, and did not initiate anaphase and telophase until correct chromosome alignment was achieved, demonstrating that the spindle checkpoint was intact. Time lapse microscopy of GFP-H2B HeLa cells revealed further mitotic abnormalities. Normally, the metaphase plate orientates so that the lateral movement of the dividing plate in anaphase is along the longest axis of the cell, parallel to the plane of the culture dish (22O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). This may involve a limited rotation of the metaphase plane in an axis perpendicular to the culture dish (Fig. 3A, Control). In cyclin A knockdown cells, the incomplete metaphase plate rotated dynamically within the cell (Fig. 3A, cyclin A siRNA, and supplemental movie S2). In some cases the condensed DNA appeared as a disk, suggesting that it had rotated to be parallel to the culture dish rather than its normal perpendicular orientation (Fig. 3A, cdc25B siRNA, 01:52). In addition, siRNA knockdown of cdc25B (supplemental Fig. 2A), the activator of G2 phase cyclin A/cdk2, produced similar rotation of the metaphase plate (Fig. 3A, cdc25B siRNA), indicating that lack of cyclin A/cdk2 activity was responsible for the rotation observed. Rotation of the metaphase plate was not simply a consequence of the extended time in mitosis, as introduction of a non-degradable form of cyclin B1 did not result in metaphase rotation during the resulting prolonged mitosis (data not shown). Confocal microscopy of cyclin A-depleted cells confirmed the rotation of the metaphase plate and spindle. Sectioning through control mitotic cells showed both spindle poles in the same section or within 3 μm in the vertical plane (supplemental Fig. S3, Control), whereas in cyclin A knockdown cells, the spindle poles were commonly found in Z-sections separated by >10 μm, and in some cells the spindle was oriented with the poles perpendicular to the plane of the culture dish (supplemental Fig. S3, Cyclin A siRNA). Quantification of mitotic abnormalities revealed that cyclin A knockdown resulted in more than 65% of the mitotic cells demonstrating rotation of the metaphase plate compared with only 15% of control cells (Fig. 3B). A consequence of this spindle rotation was that 10% of cell divisions occurred out-of-the plane of the dish in cyclin A knockdown cells compared with less than 3% out-of-plane division in controls (Fig. 3B). In the cyclin A knockdown cells the few lagging chromosomes often formed a secondary metaphase plate at an angle to the primary plate (supplemental Fig. S2B). These asymmetrical multipolar spindles were observed in 10% of cyclin A knockdown cells, whereas the controls contained 10-fold more colonies (Fig. 3C), confirming the elevated level of out-of-plane divisions observed in these cells. The mitotic spindle in cyclin A-depleted cells appeared intact although the astral microtubules appeared less directed in their contacts with the cell cortex. In controls, the astral microtubules make perpendicular contact with the cortex, whereas they appeared to fail to make direct contact with the cortex in the cyclin A-depleted cells, even where the spindle pole was in very close proximity to the cell cortex (Fig. 4A). There was also an increase in the proportion of cells where the mitotic spindle was off-centered in the cyclin A knockdown cells (55% compared with 15% in controls; Fig. 4B), most likely a direct consequence of the failure of the astral microtubules in contacting the cell cortex. Cyclin A depletion in U2OS and primary cultures of neonatal foreskin fibroblasts (NFF) with either A1 or A3 siRNA revealed similar elevated levels of off-centered spindles (supplemental Fig. S5A). This was also observed in cells undergoing anaphase (Fig. 4A), resulting in off-centered cytokinesis generating daughter cells of unequal size in 43% of these cells (Fig. 4B). To assess this in live cells, HeLa cells expressing Cherry-tubulin were examined by time lapse microscopy. Not only was it evident that the cyclin A knockdown cells spent longer in mitosis, but the mitotic spindle also moved dramatically within the cells, which frequently resulted in uneven cytokinesis (Fig. 4C). When cyclin A/cdk2 kinase activity was inhibited by the specific cdk2 inhibitor RO-09-3003 (9De Boer L. Oakes V. Beamish H. Giles N. Stevens F. Somodevilla-Torres M. Desouza C. Gabrielli B. Oncogene. 2008; 27: 4261-4268Crossref PubMed Scopus (111) Google Scholar, 23Stead E. White J. Faast R. Conn S. Goldstone S. Rathjen J. Dhingra U. Rathjen P. Walker D. Dalton S. Oncogene. 2002; 21: 8320-8333Crossref PubMed Scopus (305) Google Scholar) in G2 phase, a similar increase in the proportion of off-center mitotic spindles and uneven cytokinesis was evident (supplemental Fig. S5B). The spindle defects observed with knockdown of cyclin A or inhibition of the cyclin A/cdk2 activity are similar to those seen with disruption of the spindle-anchoring proteins, EB1 and APC. Mutation or depletion of these proteins compromise the ability of astral microtubules to make productive interactions with the cortical surface of the cell (4Green R.A. Wollman R. Kaplan K.B. Mol. Biol. Cell. 2005; 16: 4609-4622Crossref PubMed Scopus (188) Google Scholar), although cyclin A depletion had no effect on either APC or EB1 levels (data not shown). Inspection of the amino acid sequences of EB1 and APC revealed that only APC contained both consensus cdk phosphorylation sites (13 sites, indicated with an asterisks in Fig. 5A) and cyclin A binding sites (14 sites, indicated with arrowheads). Cyclin A/cdk2 co-immunoprecipitated with APC from G2/M phase cells (Fig. 5B, 8.5–9 h post-thymidine release), although this appeared to be restricted to a short period during G/M transition as there was much reduced interaction at the 7.5- and 9-h time point, even though cyclin A and APC protein levels were relatively unchanged during this time course (Fig. 5B). This restricted period of interaction was observed in multiple experiments and appeared to correspond to late G2 phase. In vitro kinase assays performed using cyclin A immunoprecipitates from synchronized HeLa cells produced a phosphorylated band that co-migrated with APC (determined by immunoblotting the kinase reaction), which was not detected in control antibody precipitates from the peak fraction (8.5 h, Fig. 5C). High levels of cyclin A-associated APC phosphorylation occurred in late G2/early M phase (Fig. 5C, 7.5 and 8.5 h), denoted by the increasing proportion of MPM2 prior to a significant increase in the proportion of cells entering mitosis, assessed using MPM-2 staining (lower panel, Fig. 5C). This corresponded to the peak of cyclin A/cdk2 activity, which preceded entry into mitosis (supplemental Fig. S6). APC mutants with truncations between codons 850 and 1450 produce mitotic phenotypes similar to cyclin A depletion (4Green R.A. Wollman R. Kaplan K.B. Mol. Biol. Cell. 2005; 16: 4609-4622Crossref PubMed Scopus (188) Google Scholar), and this region contained two potential cdk phosphorylation sites, Ser-1100 and Ser-1360. When a GST-APC fragment, APC-(1011–1470), was mixed with late G2 phase cell extracts, cyclin A/cdk2 bound to GST-APC-(1011–1470) but not to GST alone (Fig. 6A). No cyclin B was found to associate with APC-(1011–1470). In vitro kinase assay of GST-APC-(1011–1470)/cyclin A/cdk2 complex showed both the APC fragment and histone H1 were phosphorylated (Fig. 6B), and was sensitive to the cdk inhibitor roscovitine and the cdk2-specific inhibitor RO-09-3033, demonstrating that cyclin A/cdk2 was capable of binding to and phosphorylating APC-(1011–1470). The two potential cdk2 phosphorylation sites in GST-APC-(1011–1470), Ser-1100 and Ser-1360 were mutated to alanine individually (S1100A, S1360A) or together (S1100/1360A) in GST-APC-(1089–1370). Serine 1360 was the sole site phosphorylated by cyclin A/cdk2 (Fig. 6C). Cyclin B/cdk1 phosphorylates APC at a number of sites on the C terminus (24Trzepacz C. Lowy A.M. Kordich J.J. Groden J. J. Biol. Chem. 1997; 272: 21681-21684Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). GST-APC-(2133–2843), covering the C-terminal cyclin B/cdk1 phosphorylation sites, bound cyclin A and cdk2 to a similar extent as GST-APC-(1089–1370) (wild type WT and S1360A), but only APC-(1089–1370) was phosphorylated by the associated kinase (supplemental Fig. S7). APC mutants have been demonstrated to have a dominant effect over the endogenous wild type protein (4Green R.A. Wollman R. Kaplan K.B. Mol. Biol. Cell. 2005; 16: 4609-4622Crossref PubMed Scopus (188) Google Scholar, 5Caldwell C.M. Green R.A. Kaplan K.B. J. Cell Biol. 2007; 178: 1109-1120Crossref PubMed Scopus (128) Google Scholar). Therefore full-length Myc-tagged APC wild type or the S1360A mutant were transfected into HeLa cells to determine whether lack of phosphorylation of APC Ser-1360 was responsible for the mitotic defects observed with cyclin A/cdk2 depletion (Fig. 7A). Overexpression of the wild-type APC had no effect on the proportion of off-centered spindles, with 20% off-centered, which was similar to normal level detected (compare Fig. 4B, Control with 7C WT). APC S1360A mutant resulted in a significant increase in the proportion of cells
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