Ribosomal Protein S3, a New Substrate of Akt, Serves as a Signal Mediator between Neuronal Apoptosis and DNA Repair

Abstract

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.Nerve growth factor (NGF) deprivation and DNA damage can activate the intrinsic apoptotic pathway, causing cytochrome c release and caspase-dependent cell death in many cell types, including sympathetic neurons (1-5). NGF regulates neuronal apoptosis through a variety of cellular signaling mechanisms, especially the phosphoinositide 3-kinase (PI3K)/Akt pathway (6). Akt signaling promotes cell survival by phosphorylating and controlling downstream effectors in both the cytoplasm and the nucleus. For instance, Akt phosphorylates the proapoptotic Bcl-2 family member BAD (7) that belongs to the cytoplasmic apoptotic apparatus. In addition, Akt inhibits chromatin condensation during apoptosis by phosphorylating ACINUS, a nuclear factor required for apoptotic chromatin condensation (8). PI3K and Akt are predominantly located in the cytoplasm, but they are also found in the nucleus or translocate there upon stimulation by growth factors (9 -11) or DNA damage (9 -12).Ribosomal protein S3 (RPS3) is a component of the 40 S ribosomal subunit and is involved in its maturation (13). A growing body of evidence suggests that ribosomal proteins are capable of extraribosomal functions. For example, RPS3, also known as UV endonuclease III, appears to possess a general base damage endonuclease that participates in the cleavage of DNA lesions caused by UV irradiation. In addition, both RPS3 and ribosomal protein P0 have an apurinic/apyrimidinic (AP) 2 endonuclease activity functioning in DNA repair at the 3Ј side of AP sites after DNA damage (14 -16). In addition, ribosomal proteins may have apoptotic functions as follows: RPS3-a is involved in the apoptotic process in NIH3T3 cells (17), and knockdown of rpS3 leads to significant cell survival after hydrogen peroxid...

Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction

“…Various extraribosomal functions of nuclear RPS3 have been reported such as its role as an NF-B subunit in apoptosis and as an AP endonuclease in base excision repair (19,24). RPS3 localizes to nucleus, both with or without various stimulations (25,27,31). Our finding of a dose-dependent increase in nuclear localization of RPS3 under conditions of oxidative and UV stress (Fig.…”

“…We assessed the AP endonuclease activity of purified FL-RPS3, N-RPS3, and C-RPS3 on circular double-stranded depurinated substrates using a plasmid nicking assay. Depurination of plasmid substrates under acidic conditions (pH 4.5) results in generation of AP sites, and this approach has been used to assay the AP endonuclease activity of various enzymes including yeast and human RPS3 (31,32). In this assay AP sites on the substrate molecules are converted into single-stranded breaks or nicks by active AP endonucleases.…”

Phosphorylation of Ribosomal Protein S3 and Antiapoptotic TRAF2 Protein Mediates Radioresistance in Non-small Cell Lung Cancer Cells

“…Nuclear accumulation of rpS3 is reported to increase DNA repair through their endonuclease activity, thereby facilitating neuronal survival from UV radiation (30). As shown in Fig.…”

“…S2). In dividing cells, phosphorylation of rpS3 is involved in prevention of its proapoptotic function (30). As shown in Fig.…”

“…Specifically, their phosphorylation status is crucial for the control of rpS3 functions under different physiological conditions including oxidative stress (18). Also, rpS3 switches from apoptosis to DNA repair through its phosphorylation (30). Regulatory roles of rpS3 have been mainly focused on the N-terminal region, particularly the KH domain, whereas those functions for the C-terminal region have been relatively unknown (31).…”