Inflammatory cytokines induce ubiquitination and loss of the prostate suppressor protein NKX3.1

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Thesis (Ph.D.)--Georgetown University, 2008.; Includes bibliographical references. Epithelial dysplasia in the prostate is seen adjacent to foci of inflammatory atrophy. How the inflammatory process leads to cellular dysplasia and transformation is not well understood. This thesis identifies a mechanism for inflammation to cause decreased expression of a key prostate tumor suppressor protein, NKX3.1. We have found that the inflammatory cytokine TNF-alpha induced rapid ubiquitination and proteasomal degradation of NKX3.1 in vitro. Inhibition of proteasomal degradation prevented the loss of NKX3.1 and resulted in the accumulation of ubiquitin-bound NKX3.1. The C-terminus of NKX3.1 is comprised of 51 amino acids distal to the homeodomain. Deletion of the C-terminal domain of NKX3.1 resulted in prolonged protein half-life and absence of TNF-alpha-induced ubiquitination. Site-directed mutation of either or both of the two lysines (193 and 201), possible sites for ubiquitin ligation, in the C-terminus did not affect ubiquitination or protein half-life. Sequential deletion of fragments of the C-terminus identified amino acids 185-199 as the peptide region affecting NKX3.1 stability. In the C-terminal region of NKX3.1, serines 185,195, and 196 were identified as potential phosphorylation sites. Mutation of serine 185 to alanine resulted in a significant increase in protein half-life, but retained susceptibility to TNF-alpha-induced ubiquitination. In contrast, steady-state NKX3.1 turnover was unaffected by a serine 196 to alanine substitution. However, mutation of serine 196 to alanine abrogated TNF-alpha-induced ubiquitination and degradation. Serine 195 was shown to have a modulating affect and attenuated TNF-alpha-induced ubiquitination. Phosphorylation of NKX3.1 was increased in the presence of TNF-alpha, but the role of serines 185, 195, 196 was not studied. Our data provides evidence that inflammatory cytokines can reduce NKX3.1 levels. Strategies to inhibit inflammation may result in a therapeutic intervention to maintain NKX3.1 levels and slow prostate cancer development and progression.
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