Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB-1 determines its cellular location. Agency for Science, Technology and Research (A*STAR) Published version We thank the Cancer Society of New Zealand (11/07), the Maurice Wilkins Centre of New Zealand and the Dean’s Bequest Fund (Dunedin School of Medicine) for their funding support of this project. We also acknowledge the Health Research Council (HRC) of New Zealand and the Royal Society James Cook Fellowship for their ongoing support of Antony Braithwaite and his lab group. The Westmead Institute of Medical Research (WIMR) Flow Cytometry Centre, supported by the Australian NHMRC and the Cancer Institute NSW are thanked for cell sorting. Anthony Cesare and his lab are supported by grants from the NHMRC (1053195, 1106241). Srinivasaraghavan Kannan and Chandra Verma would like to thank A*STAR and National Supercomputer Centre Singapore for support. Srinivasaraghavan Kannan and Chandra Verma are founders/scientific consultants of Sinopsee Therapeutics, a biotechnology company developing molecules for therapeutic purposes; the current work does not conflict with the company.