Characterising the phosphorylation of YB-1

Abstract

The Y-Box-binding protein-1 (YB-1) is a ubiquitous and multifunctional protein involved in several cellular processes such as transcription, mRNA splicing, and translation. YB-1 has been shown to be of significance to the area of cancer research, where it has been observed to be expressed at elevated levels in~40% of invasive breast carcinomas and a number of other malignancies. Within the nucleus, it has been reported to induce the transcription of a variety of genes, including oncogenes that upregulate cell proliferation, migration, and invasion. The phosphorylation of YB-1 at serine 102 has been reported to be necessary for its nuclear translocation and transcriptional activities.In most cases, the majority of the cell’s YB-1 is present within the cytoplasm, where it is involved in processes such as mRNA translation. YB-1 translationally upregulates several EMT associated and promoting genes, potentially contributing to metastasis. Its phosphorylation at serine 102 was also reported to allow the translation of latent oncogenic transcripts that upregulate cell proliferation.«br /» «br /» The general goal of this study was to functionally characterise YB-1 phosphorylation and determine its significance on tumour malignancy. However, the specific aims of this project were: (1) To purify and characterise the phosphorylation status of cytoplasmic and nuclear YB-1 by mass spectrometry; (2) To characterise their phosphorylation profiles under normal conditions, and after treatment with a variety of genotoxic agents; (3) To determine the significance of phosphorylation on YB-1 localisation and YB-1 driven cell proliferation and migration (hallmarks of cancer), using phosphorylation-defective mutants. As detailed in this thesis, a total of 7 distinct YB-1 phosphorylation residues have now been identified from A549 lung cancer cell lysate. The contribution of these phosphorylation sites to the regulation of YB-1 localisation and functions remain unknown. Nuclear and cytoplasmic YB-1 were both individually purified and identified by mass spectrometry, and a total of 7 phosphorylation sites were identified under normal conditions, and 6 under genotoxic stress (doxorubicin, cisplatin). Experiments with the phosphorylation-defective mutants suggest that dephosphorylation in general increases YB-1 nuclear localisation in colorectal and lung cancers, with the most nuclear localisation observed with NLS-proximal phosphorylation mutants. The data also suggests that phosphorylation at the NLS proximal serine 167, 174, and 176 sites are necessary for lung cancer cells to proliferate at an accelerated rate. On the other hand, phosphorylation-defective serine 165 and serine 174 & 176 double-mutants appear to act as oncoproteins that upregulate proliferation. Migration speed was also observed to increase with the serine 174-176 double and the threonine 271 phosphorylation-defective mutants, suggesting dephosphorylation at those residues increase bone cancer cell migration. Phosphorylation-defective mutants were also found to generally increase migration persistence (i.e. towards a gradient) in bone cancer cells. These studies implicate additional YB-1 phosphorylation residues in the regulation of two major hallmarks of cancer, proliferation and metastasis, and can potentially be of valuable prognostic value in future therapeutic endeavours in cancer research.