Activation-induced nuclear translocation of RING3

Abstract

RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serum-starved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2F-regulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.

Fig. 1

Nuclear translocation of endogenous RING3 protein after serum stimulation. RING3 protein was visualised with rabbit anti-RING3 primary antibody and goat anti-rabbit IgG-FITC secondary. (A) Starved BALB cells; (B) differential interference contrast (dic); (C) serum-stimulated; (D) dic. Bars, 10 µm.

Fig. 2

Recombinant wild-type and endogenous RING3 proteins both translocate to the nucleus. Endogenous RING3 protein was visualised as in Fig. 1 and recombinant HA-tagged RING3 protein was visualised with mouse anti-HA monoclonal antibody and anti-mouse IgG-rhodamine secondary. (A) Starved, FITC (green); (B) starved, rhodamine (red); (C) merge; (D) dic; (E) serum-stimulated, FITC; (F) serum-stimulated, rhodamine; (G) merge; (H) dic. Bars, 10 µm.

Fig. 3

Recombinant K574A mutant RING3 protein also translocates to the nucleus. (A) Starved, FITC; (B) Starved, rhodamine; (C) merge; (D) dic; (E) serum-stimulated, FITC; (F) serum-stimulated, rhodamine; (G) merge; (H) dic. Bars, 10 µm.

Fig. 4

NLS peptide of RING3 is sufficient to target GFP to the nucleus. Fluorescence of BALB cells transfected with GFP constructs and visualised. (A) GFP alone; (B) dic; (C) GFP with RING3 NLS; (D) dic; (E) GFP with mutant NLS; (F) dic. Bars, 10 µm.

Fig. 5

RING3 with a mutant NLS fails to translocate to the nucleus or to transactivate. RING3 proteins were visualised as in Fig. 2. (A) Serum-stimulated, FITC; (B) serum-stimulated, rhodamine; (C) merge; (D) dic. Bar, 10 µm. (E) Transcriptional response of a luciferase construct of the dihydrofolate reductase promoter. Vector, control; RING3, wild-type recombinant RING3; HA-RING3, HAtagged wild-type recombinant RING3, HA-RING3 (NLS)^mut, mutated NLS.