Figure 1.

Strap regulation through nuclear accumulation. (A) Strap contains six TPR motifs (I–VI), with the ATM S203 phosphorylation site located in the third TPR motif and the Chk2 S221 (this study) phosphorylation site indicated. The leucine-rich NES is located in the amino-terminal region (residues 10–21). (B) U2OS cells were transfected with expression vectors (200 ng) encoding Strap or the indicated mutant derivative and, where denoted, treated with leptomycin B (LMB; 10 nM, 4 h). Immunostaining was performed with the anti-HA HA11 as described and DAPI was used to visualize nuclei. (C) Strap or the indicated mutant derivative was transfected into U2OS cells (500 ng) and thereafter left either untreated or treated with etoposide (10 μM). Extracts were prepared after 16 h and immunoblotting was performed using anti-HA antibody to detect ectopic Strap. Co-transfected β-galactosidase expression vector (200 ng) acted as a control for transfection efficiency. (D) Summary of properties of Strap mutant derivatives. The fold increase of Strap and its derivatives under DNA-damaging conditions (derived from C) is indicated in parentheses. ATM, ataxia telangiectasia mutated; Chk2, Checkpoint kinase 2; DAPI, 4,6-diamidino-2-phenylindole; HA, haemagglutinin; NES, nuclear export signal; Strap, stress responsive activator of p300; TPR, tetratricopeptide repeat; WT, wild type.