Endocrine Abstracts

Glucocorticoids (Gcs) act via the intracellular glucocorticoid receptor (GR) to regulate cellular homeostasis. Previous studies have demonstrated altered Gc sensitivity during mitosis. This reduced sensitivity can be explained only in part through the selective export of GR in mitotic cell populations. In this study ligand-independent, cell cycle-dependent effects on GR have been explored.

Mitotic cells show a marked increase in ligand-independent ser211 phosphorylation of GR, a consensus CDK/MAPK phosphorylation site. Immunofluorescent analysis using antibodies specific to ser211 phosphorylated and non-phosphorylated GR revealed a small population of hyperphosphorylated GR apparently localised to the condensed chromatin of mitotic cells. This signal was abolished using a blocking peptide specific for phospho-GR and was not evident in GR deficient U20S cells or in HeLa cells after treatment with GR-specific siRNA.

Further analysis showed the phospho-GR to be spindle-associated during anaphase, with the same pattern of distribution as that reported for mid-zone proteins such as INCENP. Disruption of microtubules did not reduce levels of phospho-GR, but altered the localisation suggesting a tethering role for the spindle. Analysis of isolated mitotic spindle extracts demonstrated localisation of GR, MAPK and CDK to the spindle. In these purified spindle samples, phospho-GR migrated into two bands with different apparent molecular mass suggesting additional post-translational modification of GR in mitotic cells.

Inhibitors of MAPK and CDK activity significantly reduced, but did not completely abolish the ligand-independent, cell cycle-dependent induction of phospho-GR. Whilst this indicates a potential role for both of these kinases in regulation of ligand-independent GR phosphorylation, it is likely that GR is also regulated by other proteins during mitosis.

Since mid-zone proteins such as INCENP are critical to successful completion of mitosis, this distinct pattern of GR distribution might suggest a previously unexpected role for GR in chromosome alignment and segregation during mitosis.