Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation

Mikael S Lindström,Yanping Zhang

doi:10.1074/jbc.m801151200

Abstract

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.

Highlights

Genomic stability, and normal ribosome biogenesis as was shown by knocking out the B23 (Npm1) gene in mice (3)
B23 Is Associated with a Ribosomal Protein-RNA Helicase Complex—To identify novel B23 interacting proteins we performed a co-IP experiment using lysates from U2OS osteosarcoma tumor cells that had been infected with adenovirus expressing Myc-tagged B23 (Ad-Myc-B23)
We found that soluble Myc-B23 protein was associated with a protein complex composed of ribosomal proteins (r-proteins) and RNA helicases, most likely representing pre-ribosomal particles at various stages of their maturation (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—U2OS osteosarcoma, H1299 lung carcinoma, and normal human fibroblasts WI38 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin in a humidified incubator. To generate stable S9-FLAG or S9-GFP cell lines, parental U2OS cells were transfected with each plasmid separately and selected in 400 ␮g/ml of G418 (Sigma). For initial screening of siRNAs stable U2OS S9-GFP cells were transfected and the knockdown was estimated by using Western blotting and cell imaging. From this screen, Ambion RPS9 siRNA number 9201 (sense 5Ј-GGAUUUCUUAGAGAGACGCTT-3Ј) was chosen as the main siRNA with the most efficient knockdown. Purification, expression, and elution of GST fusion proteins from beads were carried out according to standard protocols (Amersham Biosciences). Following primary and secondary antibody incubations in blocking buffer with extensive washings in between, the stained cells were analyzed using an Olympus IX-81 microscope with SPOT-camera and software as described. Cells on 60-mm plates were starved of methionine for 30 min with methionine-free medium and labeled with L-[methyl-3H]methionine

RESULTS

B23 Increases S9 Steady-state

DISCUSSION