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X.), and by NSFC fund 31271446 (J. protein levels by siRNA treatment or the S20A mutation hampers the phosphorylation of vimentin at Ser-71, resulting in a failure of vimentin bridge disassembly. Our results not only exemplified another crosstalk between and and and kinase (IVK) assays. Commercially available GST-Chk1 was incubated with GST-OGT or GST-OGT(S20A) proteins and subject to IVK assays. Chk1 phosphorylated OGT efficiently but not the OGT(S20A) mutant (Fig. 2fit the consensus motif. C, kinase (IVK) assays were carried out using recombinant Chk1, OGT-WT, and OGT(S20A) mutant proteins. The IVK products were then blotted with antiCpSer-20 antibodies. and and and are in supplemental Figs. S2 and S3, respectively. OGTCpSer-20 localizes to the midbody We then addressed the question concerning in which intracellular compartments the phosphorylated OGT resides. As shown in Fig. 4both of Dipsacoside B which efficiently depleted OGT protein levels (Fig. 4the midbody localization of pSer-20 significantly decreased (Fig. 4, and to deplete Dipsacoside B Chk1 (Fig. 4(Fig. 4treatments (two different oligos), and then extracts were immunoprecipitated with antibodies indicated. treatment (two different oligos) were stained with anti-OGT, pSer-20, and -tubulin antibodies, together with DAPI. test (treatment, and then extracts were immunoblotted with antibodies indicated. in (in (in (= 0.0001). OGT(S20A) attenuates vimentin O-GlcNAcylation and phosphorylation Previous investigations have implicated interplay of and then synchronized in the cytokinetic stage (Fig. 5oligos or left untreated, then synchronized in the cytokinetic phase as described in Experimental Procedures. The total lysates were subject to IB with the antibodies indicated. Numbers indicate levels of RL2 and vimentinCpSer-71, respectively. The data are representative of three independent experiments. (two independent oligos), then subject to immunostaining with anti-vimentin antibodies and DAPI. cells. oligos were quantitated for anaphase arrest plus multinucleated cells, indicating defects in cytokinesis. At least 100 cells were counted. The results are mean S.D. of three independent experiments. * indicated significant differences from WT (cells (Fig. 5cells (Fig. 5cells (Fig. 5treatment (Fig. 6and plasmids, and then treated with control siRNA or sias indicated. The lysates were synchronized in the cytokinetic stage and subject to IB using the antibodies indicated. Numbers indicate levels of vimentinCpSer-71. The data are representative of three independent experiments. were subject to immunostaining with anti-vimentin antibodies and DAPI. were quantitated for anaphase arrest plus multinucleated cells, indicating defects in cytokinesis. At least 100 cells were counted. The results are mean S.D. of three independent experiments. * indicates significant differences from WT (in synchronized cytokinetic cells and examined biochemically whether vimentinCpSer-71 was affected (Fig. 7and then synchronized in cytokinesis and examined with the indicated antibodies by IB. Numbers indicate levels of vimentinCpSer-71. The data are cIAP2 representative of three independent experiments. cells. = 0.02). and suppressed the cellular migration and invasion, whereas Thiamet-G (OGA inhibitor) bolstered it. More importantly, Thiamet-G boosted RhoA activity and the phosphorylation of Rho kinase substrates, Dipsacoside B whereas sidampened RhoA activity and subsequent Rho kinase substrate phosphorylation (24). Collectively, sequences were amplified by PCR and cloned into pcDNA3.0C3HA, resulting in pcDNA-3HA-OGT. Myc-OGT and GST-OGT plasmids were described previously (30), and were gifts from Drs. Huadong Pei and Xiaochun Yu. OGT(S20A) mutants were generated using specific primers (sequences available upon request) following the manufacturer’s instructions (QuikChange II, Stratagene). Cell synchronization Protocols to synchronize cells in the cytokinetic phase were described before (31). Briefly, cell cultures were first blocked by double thymidine, and collected 9 h after releasing from the second thymidine block. Transfections HeLa cells were transfected twice with a 24-h interval using Oligofectamine (Invitrogen) according to the manufacturer’s instructions. Transfectants were used for further experiments 24 h after the second transfection. All small interfering RNA (siRNA) oligonucleotide duplexes were purchased from Dharmacon. The Dipsacoside B control siRNA oligonucleotide duplex was CONTROLsi, CGUACGCGGAAUACUUCGAdTdT; si1#, GCAGUAGCUUGGAGUAAUC dTdT; si3#, GCACGGCUCUGAAACUUAA dTdT; sikinase.