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• W2040892143 abstract "Microtubule dynamics is essential for many vital cellular processes such as morphogenesis and motility. Protein kinase CK2 is a ubiquitous protein kinase that is involved in diverse cellular functions. CK2 holoenzyme is composed of two catalytic α or α′ subunits and two regulatory β subunits. We show that the α subunit of CK2 binds directly to both microtubules and tubulin heterodimers. CK2 holoenzyme but neither of its individual subunits exhibited a potent effect of inducing microtubule assembly and bundling. Moreover, the polymerized microtubules were strongly stabilized by CK2 against cold-induced depolymerization. Interestingly, the kinase activity of CK2 is not required for its microtubule-assembling and stabilizing function because a kinase-inactive mutant of CK2 displayed the same microtubule-assembling activity as the wild-type protein. Knockdown of CK2α/α′ in cultured cells by RNA interference dramatically destabilized their microtubule networks, and the destabilized microtubules were readily destructed by colchicine at a very low concentration. Further, over-expression of chicken CK2α or its kinaseinactive mutant in the endogenous CK2α/α′-depleted cells fully restored the microtubule resistance to the low dose of colchicine. Taken together, CK2 is a microtubule-associated protein that confers microtubule stability in a phosphorylation-independent manner. Microtubule dynamics is essential for many vital cellular processes such as morphogenesis and motility. Protein kinase CK2 is a ubiquitous protein kinase that is involved in diverse cellular functions. CK2 holoenzyme is composed of two catalytic α or α′ subunits and two regulatory β subunits. We show that the α subunit of CK2 binds directly to both microtubules and tubulin heterodimers. CK2 holoenzyme but neither of its individual subunits exhibited a potent effect of inducing microtubule assembly and bundling. Moreover, the polymerized microtubules were strongly stabilized by CK2 against cold-induced depolymerization. Interestingly, the kinase activity of CK2 is not required for its microtubule-assembling and stabilizing function because a kinase-inactive mutant of CK2 displayed the same microtubule-assembling activity as the wild-type protein. Knockdown of CK2α/α′ in cultured cells by RNA interference dramatically destabilized their microtubule networks, and the destabilized microtubules were readily destructed by colchicine at a very low concentration. Further, over-expression of chicken CK2α or its kinaseinactive mutant in the endogenous CK2α/α′-depleted cells fully restored the microtubule resistance to the low dose of colchicine. Taken together, CK2 is a microtubule-associated protein that confers microtubule stability in a phosphorylation-independent manner. Protein kinase CK2 (formerly known as casein kinase 2) is ubiquitously expressed and highly conserved in eukaryotic cells (1Allende J.E. Allende C.C. FASEB J. 1995; 9: 313-323Crossref PubMed Scopus (586) Google Scholar, 2Blanquet P.R. Prog. Neurobiol. 2000; 60: 211-246Crossref PubMed Scopus (142) Google Scholar, 3Litchfield D.W. Biochem. J. 2003; 369: 1-15Crossref PubMed Scopus (1017) Google Scholar, 4Pinna L.A. Meggio F. Prog. Cell Cycle Res. 1997; 3: 77-97Crossref PubMed Scopus (312) Google Scholar). It comprises two catalytic α or α′ subunits and two regulatory β subunits to form a heterotetrameric structure in which the two β subunits dimerize to link the two α or α′ subunits (5Niefind K. Guerra B. Ermakowa I. Issinger O.G. EMBO J. 2001; 20: 5320-5331Crossref PubMed Scopus (331) Google Scholar). As a protein serine/threonine kinase, CK2 has a very broad phosphorylation spectrum, and over 300 protein substrates of CK2 have been identified to date (6Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1098) Google Scholar). A number of studies have indicated that CK2 is involved in a wide variety of cellular processes including cell cycle, apoptosis, transcriptional regulation, and signal transduction (1Allende J.E. Allende C.C. FASEB J. 1995; 9: 313-323Crossref PubMed Scopus (586) Google Scholar, 3Litchfield D.W. Biochem. J. 2003; 369: 1-15Crossref PubMed Scopus (1017) Google Scholar, 6Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1098) Google Scholar). CK2 is instrumental and necessary for promoting cell survival (3Litchfield D.W. Biochem. J. 2003; 369: 1-15Crossref PubMed Scopus (1017) Google Scholar, 7Ahmed K. Gerber D.A. Cochet C. Trends Cell Biol. 2002; 12: 226-230Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Disruption of genes encoding both of the catalytic subunits of CK2 is synthetic lethal in fission yeast (8Padmanabha R. Chen-Wu J.L. Hanna D.E. Glover C.V. Mol. Cell Biol. 1990; 10: 4089-4099Crossref PubMed Scopus (305) Google Scholar, 9Rethinaswamy A. Birnbaum M.J. Glover C.V. J. Biol. Chem. 1998; 273: 5869-5877Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Similarly, it is embryonic lethal when CK2β is knocked down in Caenorhabditis elegans by RNA interference or in mice by gene disruption, reminiscent of an essential role of CK2β during embryonic development and organogenesis (10Buchou T. Vernet M. Blond O. Jensen H.H. Pointu H. Olsen B.B. Cochet C. Issinger O.G. Boldyreff B. Mol. Cell Biol. 2003; 23: 908-915Crossref PubMed Scopus (217) Google Scholar, 11Fraser A.G. Kamath R.S. Zipperlen P. Martinez-Campos M. Sohrmann M. Ahringer J. Nature. 2000; 408: 325-330Crossref PubMed Scopus (1360) Google Scholar). Hence, production of both the α and β subunits of CK2 appears to be mandatory for cell viability.A few lines of evidence have lead to implication that CK2 might be involved in the regulation of microtubule cytoskeleton reorganization (12Diaz-Nido J. Serrano L. Mendez E. Avila J. J. Cell Biol. 1988; 106: 2057-2065Crossref PubMed Scopus (136) Google Scholar, 13Serrano L. Diaz-Nido J. Wandosell F. Avila J. J. Cell Biol. 1987; 105: 1731-1739Crossref PubMed Scopus (86) Google Scholar, 14Serrano L. Hernandez M.A. Diaz-Nido J. Avila J. Exp. Cell Res. 1989; 181: 263-272Crossref PubMed Scopus (55) Google Scholar). CK2 was localized to microtubule structures such as the mitotic spindle of dividing cells and was found to associate with the cold-stable fraction of microtubules from the rat brain (14Serrano L. Hernandez M.A. Diaz-Nido J. Avila J. Exp. Cell Res. 1989; 181: 263-272Crossref PubMed Scopus (55) Google Scholar, 15Diaz-Nido J. Avila J. Second Messengers Phosphoproteins. 1992; 14: 39-53PubMed Google Scholar). More recently, the α and α′ subunits were shown to bind tubulin in a far Western assay (16Faust M. Schuster N. Montenarh M. FEBS Lett. 1999; 462: 51-56Crossref PubMed Scopus (71) Google Scholar). Further, CK2 is able to phosphorylate a number of microtubule elements, including MAP1B and a neuron-specific β-tubulin isotype (6Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1098) Google Scholar). The phosphorylation of MAP1B was proposed to facilitate the microtubule association of MAP1B and thereby microtubule assembly, whereas the physiological role of the β-tubulin isotype phosphorylation is still unclear (12Diaz-Nido J. Serrano L. Mendez E. Avila J. J. Cell Biol. 1988; 106: 2057-2065Crossref PubMed Scopus (136) Google Scholar, 17Diaz-Nido J. Serrano L. Lopez-Otin C. Vandekerckhove J. Avila J. J. Biol. Chem. 1990; 265: 13949-13954Abstract Full Text PDF PubMed Google Scholar). Despite these findings, the direct correlation of CK2 and microtubule stability has not been established.In the present study, we have investigated the physical association of CK2 with microtubules and the direct effect of CK2 on microtubule dynamics. Our results show that CK2 is a microtubule-associated protein (MAP) 1The abbreviations used are: MAPmicrotubule-associated proteinGSTglutathione S-transferasePBSphosphate-buffered salinesiRNAsmall-interfering RNAPIPES1,4-piperazinediethanesulfonic acid.1The abbreviations used are: MAPmicrotubule-associated proteinGSTglutathione S-transferasePBSphosphate-buffered salinesiRNAsmall-interfering RNAPIPES1,4-piperazinediethanesulfonic acid. that induces microtubule assembly and bundling in vitro. CK2-polymerized microtubules appear stable under cold treatment. In cultured cells, knockdown of CK2α/α′ has a severe effect on microtubule stability, which implies that CK2 mediates microtubule integrity in vivo. Moreover, a kinase-inactive mutant of CK2 displayed the same microtubule polymerizing and stabilizing activity in vitro and in vivo. Thus, the microtubule assembling and stabilizing action of CK2 is independent of its kinase function.EXPERIMENTAL PROCEDURESPlasmid Constructions—The coding sequences of chicken CK2α and its kinase-inactive mutant (CK2αK68A) were subcloned into pGEX4T (Amersham Biosciences), pET32 (Novagen), and pDneo-Myc (18Tang B.L. Ong Y.S. Huang B. Wei S. Wong E.T. Qi R. Horstmann H. Hong W. J. Biol. Chem. 2001; 276: 40008-40017Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The full-length sequence of human CK2β was cloned by a reverse transcription polymerase chain reaction and inserted into pQE30 (Qiagen).Protein Binding Assay—Proteins tagged with GST or His6 were bacterially expressed and prepared as described previously (19Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To test tubulin binding, GSH-Sepharose beads (Amersham Biosciences) prebound with GST, GST-CK2α, or the complex of GST-CK2α/His-CK2β were incubated with purified tubulin (>99% pure and MAP-free, Cytoskeleton) for 1 h at 4 °C. After being extensively washed with binding buffer (20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 20 mm MgCl2, 1 mm dithiothreitol, and 0.1% Nonidet P-40), the beads were boiled in SDS-PAGE sample buffer and analyzed by immunoblotting. Antibodies against α- and β-tubulin were from Sigma. The binding of His-tagged proteins with tubulin was performed with nickel-nitrilotriacetic acid beads (Ni-NTA, Qiagen) in binding buffer without dithiothreitol. In the microtubule binding assay, microtubules, which were pre-assembled using taxol in PEM buffer (80 mm PIPES, pH 6.8, 1 mm MgCl2, 1 mm EGTA) supplemented with 1 mm GTP, were incubated with the indicated proteins. The samples were subsequently loaded onto a buffered cushion (50% glycerol in PEM buffer) and centrifuged to spin down the microtubules and associated proteins. The pellet and the supernatant were analyzed by immunoblotting.Microtubule Assembly—Microtubules were assembled in vitro from the purified MAP-free tubulin at 2 mg/ml in PEM buffer supplemented with 1 mm GTP at 35 °C, and the turbidity of the solutions was monitored at 340 nm (20Gaskin F. Methods Enzymol. 1982; 85: 433-439Crossref PubMed Scopus (26) Google Scholar). CK2 was added at various amounts as indicated to promote the assembly. To visualize assembled microtubules, tubulin and rhodamine-labeled tubulin (Cytoskeleton) at the ratio of 7:1 were used in the polymerization (21Belmont L.D. Hyman A.A. Sawin K.E. Mitchison T.J. Cell. 1990; 62: 579-589Abstract Full Text PDF PubMed Scopus (344) Google Scholar). Microtubules were fixed with 0.5% gluteraldehyde and visualized by fluorescence microscopy.Differential Tubulin Extraction from Intact Cells—Differential extraction of tubulin heterodimers and polymers from cells was performed using a protocol described previously (22Lieuvin A. Labbe J.C. Doree M. Job D. J. Cell Biol. 1994; 124: 985-996Crossref PubMed Scopus (100) Google Scholar). Briefly, cultured cells were lysed with the microtubule-stabilizing buffer (80 mm PIPES, pH 6.8, 1 mm MgCl2, 1 mm EGTA, 0.5% Triton X-100, 10% glycerol, and Roche protease inhibitor mixture), which was prewarmed to 35 °C, to extract cytosolic soluble tubulin heterodimers and preserve microtubules (assembled insoluble tubulin polymers). The extract was cleared by centrifugation and the supernatant designated as the free tubulin fraction. After a brief washing with the microtubule-stabilizing buffer, the pellet was extracted in the microtubule-destabilizing buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100, 10 mm CaCl2, and Roche protease inhibitor mixture). The extract was clarified by centrifugation to yield the polymerized tubulin fraction. Both fractions were analyzed by immunoblotting, and each band on the blots was quantitated using a Bio-Rad GS-700 imaging densitometer and analyzed with the Multi-Analyst, version 1.0.1, program (Bio-Rad).Cell Culture, Transfection, and Immunofluorescence—COS-7, HeLa and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The siRNA sequence designed for human CK2α/α′ is 5′-CCAGCUGGUAGUCAUCUUGUU-3′, which has a few discrepancies with the corresponding sequence of chicken CK2α/α′.20 μm CK2α/α′ siRNA or a scrambled siRNA sequence was applied into the transfection using a TransIT-TKO transfection reagent (Mirus). Simultaneous transfection of siRNA and plasmid DNA was done using TransIT-TKO and LipofectAMINE (Invitrogen) concurrently. After transfection, the cells were cultured for 24 h before treatment with 0.2 μm colchicine (Sigma) for 3 h. The cells were subjected to differential extraction of free and polymerized tubulin or to immunostaining. For immunofluorescence, the cells were fixed in PBS containing 4% paraformaldehyde and then permeabilized in PBS containing 0.2% Triton X-100. After a blocking wash with 10% goat serum and 0.1% Triton X-100 in PBS, immunostaining was performed with antibodies as indicated. CK2α- and CK2β-specific antibodies were from Santa Cruz Biotechnology. The secondary antibodies are Fluor594 goat anti-mouse IgG and Fluor488 donkey anti-goat IgG (Molecular Probes). The cells were then washed in PBS, mounted, and photographed on an MRC-1024 laser scanning confocal microscope (Bio-Rad).RESULTSCK2 Forms a Direct Complex with Microtubules—The direct association of CK2 and microtubules was probed by a series of binding assays using recombinant CK2 and purified MAP-free tubulin as well as pre-assembled microtubules. The α/β heterodimer of tubulin was found to associate with the catalytic α subunit as well as the holoenzyme of CK2 (Fig. 1A). CK2β alone did not result in the pull-down of any tubulin (Fig. 1B), which is in agreement with a previous observation using far Western blotting (16Faust M. Schuster N. Montenarh M. FEBS Lett. 1999; 462: 51-56Crossref PubMed Scopus (71) Google Scholar). To verify the microtubule association of CK2, taxol-assembled microtubules were incubated with CK2 holoenzyme or its individual subunit proteins. The microtubules were then spun down to test whether these proteins co-precipitated with the microtubules. Consistently, CK2α and the holoenzyme of CK2 were found to associate with the microtubule pellet, whereas CK2β and GST, as a control protein, failed to co-precipitate with the microtubules (Fig. 1C), indicating that the CK2 holoenzyme associates with microtubules at a high affinity through CK2α.Cellular localization of CK2 to microtubule networks was revealed by immunofluorescent staining of cultured COS-7 cells. Microscopic imaging of endogenous CK2α and CK2β displayed a clearly defined positioning with the microtubule network, particularly in the cell periphery (Fig. 2A). As confirmation, pools of tubulin existing as free heterodimers or polymers (microtubules) were differentially extracted from the cultured cells to examine the distribution of CK2 (22Lieuvin A. Labbe J.C. Doree M. Job D. J. Cell Biol. 1994; 124: 985-996Crossref PubMed Scopus (100) Google Scholar). Both CK2α and CK2β appeared in the microtubule fraction as well as the fraction of free tubulin heterodimers, although there appeared to be more CK2β in the microtubule fraction (Fig. 2B). Taken together with the results from the in vitro binding assays, this provides evidence of the direct association of CK2 with cellular microtubules.Fig. 2Cellular localization of CK2α and CK2β to microtubules.A, COS-7 cells were immunostained for confocal microscopic analysis. Top row, double staining of CK2α and β-tubulin; bottom row, CK2β and β-tubulin. B, soluble tubulin heterodimers (free tubulin) and microtubules (polymerized tubulin) were differentially extracted from HeLa cells. Both fractions as well as the total cell lysate (TCL) were analyzed by immunoblotting using antibodies as indicated. The histogram shows the relative amounts of CK2α and CK2β in the free and polymerized tubulin fractions. These data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CK2 Induces Microtubule Polymerization—We next investigated whether CK2 has any effect on microtubule dynamics by using an in vitro assay of microtubule assembly from purified MAP-free tubulin (20Gaskin F. Methods Enzymol. 1982; 85: 433-439Crossref PubMed Scopus (26) Google Scholar). During the assay, the turbidity change of the solution was measured as tubulin polymerizes or depolymerizes. In the absence of CK2, there was minimal polymerization of tubulin even after a prolonged incubation (Fig. 3, A and B). The addition of CK2 at a ratio of 1:240 to tubulin resulted in substantial polymerization of tubulin into microtubules (Fig. 3, A and B). Clearly, both the rate and extent of polymerization were dramatically enhanced by CK2. When the amount of CK2 was increased, tubulin polymerization was increased in a dose-dependent manner (Fig. 3, A and B). To verify the microtubule formation, rhodamine-labeled tubulin was applied into the polymerization experiments for direct visualization of the assembled microtubules by fluorescence microscopy (21Belmont L.D. Hyman A.A. Sawin K.E. Mitchison T.J. Cell. 1990; 62: 579-589Abstract Full Text PDF PubMed Scopus (344) Google Scholar). As shown in Fig. 3C, microtubule filaments and bundles were readily observed with the CK2-incubated tubulin, whereas the incubation of tubulin without CK2 showed no obvious microtubule formation. Therefore, we have found that CK2, in addition to showing high affinity binding to tubulin and microtubules, induces the assembly of tubulin into microtubules. Moreover, CK2 appeared to cause microtubule bundling, suggesting a strong stabilizing effect on the microtubules.Fig. 3Effect of CK2 on microtubule assembly.A, the turbidimetric assay of tubulin polymerization. Microtubule assembly from purified MAP-free tubulin was carried out in the presence of the CK2 holoenzyme at various concentrations (molar ratios to tubulin). The concentration of tubulin was constant in each assay at 2 mg/ml. B, histogram of the microtubule assembly at various amounts of CK2. The assembly assay was performed as described in A for 30 min. The data shown are representative of three separate experiments. C, fluorescent imaging of microtubules polymerized from a mixture of rhodamine-labeled and unlabeled tubulin (7:1). The tubulin concentration is 2 mg/ml, and the CK2 concentration is 62 μg/ml. The arrows point to microtubule bundles in the CK2-polymerized sample.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CK2 holoenzyme is a tetrameric complex of two α or α′ subunits and two β subunits (5Niefind K. Guerra B. Ermakowa I. Issinger O.G. EMBO J. 2001; 20: 5320-5331Crossref PubMed Scopus (331) Google Scholar). Given that observation that CK2α of the holoenzyme interacts with microtubules, we explored whether the microtubule assembling function of CK2 is restricted to the holoenzyme by applying the α and β subunits of CK2 individually into the microtubule assembly assay. In contrast to the holoenzyme, when either the α or β subunit was tested, there was minimal polymerization of tubulin even after a prolonged incubation (Fig. 4). The CK2α- and CK2β-polymerized samples had no marked difference from the background tubulin polymerization, which was shown in the GST-incubated sample. Thus, only CK2 holoenzyme, but not each of the individual subunits, has the ability to induce microtubule assembly even though CK2α has shown microtubule binding activity.Fig. 4Microtubule assembly can be induced by the CK2 holoenzyme but not its individual subunits. GST, GST-CK2α, and His-CK2β were applied as indicated at 0.1 mg/ml in the microtubule assembly assay. As a control, the CK2 holoenzyme reconstituted from the same amount of GST-CK2α and His-CK2β as described under “Experimental Procedures” was applied. Microtubule assembly was performed at 2 mg/ml tubulin as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)CK2 has been known to catalyze phosphorylation of a neural isoform of β-tubulin and some of the MAPs, raising the possibility that it may affect microtubule dynamics through a kinase reaction (12Diaz-Nido J. Serrano L. Mendez E. Avila J. J. Cell Biol. 1988; 106: 2057-2065Crossref PubMed Scopus (136) Google Scholar, 17Diaz-Nido J. Serrano L. Lopez-Otin C. Vandekerckhove J. Avila J. J. Biol. Chem. 1990; 265: 13949-13954Abstract Full Text PDF PubMed Google Scholar). Although ATP was not present in the in vitro microtubule assembly assay, CK2 is capable of utilizing either ATP or GTP as the phosphate donor in its phosphorylating reactions (23Niefind K. Putter M. Guerra B. Issinger O.G. Schomburg D. Nat. Struct. Biol. 1999; 6: 1100-1103Crossref PubMed Scopus (160) Google Scholar). We designed an experiment to assess the role of CK2 kinase activity in microtubule assembly. A kinase-inactive holoenzyme of CK2, in which CK2α was replaced with the kinase-inactive mutant CK2αK68A, was tested in the microtubule assembly assay. Fig. 5 shows that the kinase-inactive CK2 conferred the same microtubule polymerizing activity as the wild-type enzyme, indicating that the microtubule assembly entity of CK2 is independent of its kinase activity and phosphorylation of any microtubule proteins.Fig. 5The kinase activity of CK2 is not required for its function to induce microtubule assembly. The wild-type CK2 enzyme (GST-CK2α/His-CK2β) and the kinase-inactive enzyme (GST-CK2αK68A/His-CK2β) were applied as indicated at 0.1 mg/ml in the microtubule assembly assay. GST-CK2αK68A and GST were also tested at the same amount. Microtubule assembly was performed with 2 mg/ml tubulin as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Microtubules from brains can be separated into two pools, namely “cold labile” and “cold stable,” according to whether they are resistant to cold treatment for microtubule disassembly (24Webb B.C. Wilson L. Biochemistry. 1980; 19: 1993-2001Crossref PubMed Scopus (81) Google Scholar). It has been found that CK2 is enriched in the coldstable fraction of the microtubule preparation from rat brain (14Serrano L. Hernandez M.A. Diaz-Nido J. Avila J. Exp. Cell Res. 1989; 181: 263-272Crossref PubMed Scopus (55) Google Scholar). This observation, together with our findings that CK2 associates with microtubules to promote microtubule assembly, prompted us to explore the possibility that CK2 may contribute to the cold stability of microtubules. To test this likelihood, CK2-polymerized microtubules were incubated on ice, and the turbidity change was monitored. As a comparison, tau-polymerized microtubules were treated under the same condition, given the fact that tau does not confer cold stability to microtubules (25Baas P.W. Pienkowski T.P. Cimbalnik K.A. Toyama K. Bakalis S. Ahmad F.J. Kosik K.S. J. Cell Sci. 1994; 107: 135-143PubMed Google Scholar). As expected, the tau-polymerized sample was depolymerized almost completely within a few minutes (Fig. 6). However, the turbidity of the CK2-polymerized sample was only marginally reduced even after a prolonged cold incubation (Fig. 6), indicating that CK2 functions to stabilize microtubules against cold-induced disassembly.Fig. 6CK2 confers cold stability to microtubules. Microtubules were polymerized with 0.05 mg/ml CK2 or 0.16 mg/ml tau protein for 30 min at 35 °C, where they attained similar turbidity measurements. The microtubule samples were then incubated on ice, and the turbidity measurement was begun. Absorbance was expressed as a percentage of the measurement when ice incubation was started.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CK2 Stabilizes Microtubules in Vivo—To evaluate the role of CK2 in microtubule dynamics in vivo, we knocked down CK2α/α′ in HeLa cells by gene silencing using a siRNA duplex derived from the human sequence of CK2α/α′ (26Sayed M. Pelech S. Wong C. Marotta A. Salh B. Oncogene. 2001; 20: 6994-7005Crossref PubMed Scopus (52) Google Scholar, 27Ulloa L. Diaz-Nido J. Avila J. EMBO J. 1993; 12: 1633-1640Crossref PubMed Scopus (156) Google Scholar). As shown by the CK2α immunoblot, the introduction of CK2α/α′ siRNA into the cells led to a dramatic decrease of the CK2α/α′ proteins to a minimal cellular level (Fig. 7A). To assess the knockdown effect on microtubules, the amount of cellular microtubules (assembled insoluble tubulin polymers) was determined using the differential extraction method and immunoblotting (22Lieuvin A. Labbe J.C. Doree M. Job D. J. Cell Biol. 1994; 124: 985-996Crossref PubMed Scopus (100) Google Scholar). In addition, the integrity of the cellular microtubule network was examined by immunofluorescent staining and confocal microscopy. The knockdown of CK2α/α′ significantly reduced the cellular content of microtubules (Fig. 7, A and B), suggesting CK2 as one of the factors in stabilizing microtubules in vivo. We further assessed microtubule stability using colchicine, which is a microtubule-disrupting agent. When colchicine was applied at a low concentration (0.2 μm) onto the cells that were transfected with a scrambled siRNA sequence, most of the microtubule structure remained intact (Fig. 7, A and B). However, such a low dose of colchicine caused severe disruption of the microtubule structure in the CK2α/α′-depleted cells where the microtubule networks were collapsing toward the perinuclear membrane (Fig. 7B); almost negligible amount of microtubules was extracted from these cells (Fig. 7A). Apparently, the removal of CK2α had a strong effect on cellular microtubule architecture, rendering it very unstable. As a result, it was readily destructed by colchicine at a very low concentration.Fig. 7CK2 stabilizes microtubules in vivo.A, HeLa cells were introduced with siRNA of human CK2α/α′ or a scrambled sequence. Knockdown of CK2α was monitored by anti-CK2α immunoblotting. The cells were subsequently treated with 0.2 μm colchicine or its solvent. Tubulin in the form of polymers (microtubules) was extracted from the cells for β-tubulin immunoblotting. The histogram reflects the relative amounts of microtubules extracted from the cells as compared with the control, which is the sample transfected with the scrambled siRNA sequence and treated without colchicine. The data are representative of three separate experiments. B, cells in the experiments described in A were fixed and stained with the β-tubulin antibody for confocal microscopic imaging. C, expression of the wild-type or the kinase-inactive mutant of chicken CK2α restored microtubule stability against colchicine treatment in CK2α/α′-depleted cells. Prior to treatment with colchicine (0.2 μm), 293T cells were double transfected with siRNA of human CK2α/α′ and one of the following expression constructs: chicken CK2α, the kinase-inactive mutant of chicken CK2α (CK2αK68A), or the empty vector. Expression of Myc-tagged chicken CK2α and CK2αK68A was detected by anti-Myc immunoblotting of the cell lysates. Microtubules were extracted using the differential extraction method (see “Experimental Procedures”) for anti-β-tubulin immunoblotting. Representative results of three separate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further substantiate the microtubule stabilizing function of CK2, we tested whether microtubule stability could be restored by expression of chicken CK2α in endogenous CK2α/α′-depleted cells. As observed with the HeLa cells, knockdown of CK2α/α′ in cultured human 293T fibroblasts using siRNA strongly destabilized the microtubule network, resulting in almost complete disruption of the microtubules by colchicine at 0.2 μm (Fig. 7C). When chicken CK2α was expressed in the 293T cells in which endogenous CK2α/α′ was knocked down, the cellular microtubules completely retained their integrity against the colchicine-induced disruption (Fig. 7C). More interestingly, when the expression was performed using the kinase-inactive mutant CK2αK68A, it exhibited the same effect as wild-type CK2α in rescuing microtubules from colchicine treatment (Fig. 7C). These data demonstrate that CK2 is an important mediator of cellular microtubule stability and exerts its eff" @default.
• W2040892143 created "2016-06-24" @default.
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• W2040892143 date "2004-02-01" @default.
• W2040892143 modified "2023-10-01" @default.
• W2040892143 title "Direct Regulation of Microtubule Dynamics by Protein Kinase CK2" @default.
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• W2040892143 doi "https://doi.org/10.1074/jbc.m310563200" @default.
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