Checkpoint kinase 1–induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis

Abstract

Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified linked β--acetylglucosamine (-GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an sibased approach. Lastly, expression of plasmids resistant to si efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis.

Vimentin and Non-Muscle Myosin IIA are Members of the Neural Precursor Cell Expressed Developmentally Down-Regulated 9 (NEDD9) Interactome in Head and Neck Squamous Cell Carcinoma Cells

“…Vimentin phosphorylation regulates a number of cellular functions. For example, Ser71 phosphorylation was reported to stimulate vimentin filament formation during cytokinesis [59] . The NEDD9 paralog, p130Cas, was reported to be a vimentin interacting protein that is displaced from vimentin upon phosphorylation of Ser56 [46] , [47] .…”

DNA damage response proteins regulating mitotic cell division: double agents preserving genome stability

“…Chk1 inhibition by the small‐molecule inhibitor UCN‐01 does not reduce Aurora B‐S331 phosphorylation at the midbody compared with control cells, indicating that Chk1 does not regulate abscission through Aurora B‐S331 phosphorylation; instead, the Cdc‐like kinases 1, 2, and 4 are required for Aurora B‐S331 phosphorylation and complete Aurora B activation in late cytokinesis . Because Chk1‐deficient HeLa cells exhibit increased frequency of intermediate filament (vimentin) bridges compared with controls in late cytokinesis , one possibility is that Chk1 prevents furrow regression by promoting vimentin severing through an incompletely understood mechanism (Fig. A).…”

Crosstalk between phosphorylation and O‐GlcNAcylation: friend or foe

“…Kaasik et al [82] showed that GSK3b can phosphorylate OGT at Ser3 and/or Ser4, whereby these phosphorylations enhance OGT activity. Recently, Li et al [83] showed that Checkpoint kinase 1 can phosphorylate OGT on Ser20, which seemingly improves OGT stability. In addition, AMPK was found to phosphorylate OGT on Thr444, which induced changes in OGT substrate specificity [34,84].…”