Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase.

Aadhya Tiwari,Eva Maier,Daniel Zips,Simone Rebholz,Christine Sers,Mahmoud Toulany,Mozhgan Dehghan Harati,H Rodemann

doi:10.3390/ijms19082441

Abstract

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.

Highlights

Y-box binding protein-1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily encoded by the YBX1 gene
YB-1 Is Not Translocated to the Nucleus after Irradiation
We demonstrated that exposure to clinically relevant doses of ionizing radiation induces phosphorylation of YB-1 in KRAS wild-type (KRASwt) but not in KRAS mutated (KRASmut) breast cancer cells [18]. It is not known whether acute phosphorylation of YB-1, such as occurs after irradiation, leads to nuclear accumulation of YB-1

Summary

Introduction

Y-box binding protein-1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily encoded by the YBX1 gene. A wide variety of cellular functions, including DNA repair, gene transcription, mRNA splicing, translation, drug resistance, and stress responses to extracellular signals, are regulated by YB-1 [3,4]. To exert the described functions, YB-1 must be phosphorylated at serine 102 (Ser-102) within the CSD. Independent of the mechanism of DSB repair by YB-1, YB-1 as a DNA damage response element needs to be present in the nucleus immediately after irradiation. This can occur through IR-induced translocation of phosphorylated YB-1 from the cytoplasm to the nucleus. P90 ribosomal S6 kinase (RSK) translocates to the nucleus and stimulates already-existing YB-1 in the nucleus

YB-1 Is Not Translocated to the Nucleus after Irradiation

Discussion

Antibodies and Reagents

Cellular Fractionation and Immunoblotting Analysis

Densitometry and Statistics