INVESTIGATING THE NEDD4-MEDIATED UBIQUITINATION OF PMEPA1, AND ITS POTENTIAL ROLE IN THE REGULATION OF THE ANDROGEN RECEPTOR AS PART OF THE STEROID RESPONSE PATHWAY IN PROSTATIC CANCER

Abstract

Ubiquitination is an extremely important post-translational modification, regulating a wide variety of cellular processes including proteasomal degradation, subcellular targeting, endocytosis and DNA repair. The HECT class of E3 ligases catalyse the final step of ubiquitin conjugation to the substrate; the Nedd4 family make up 9 members of this class in humans, and are implicated in pathologies ranging from congenital ion channel misregulation to cancer, via the TGF-ß signalling pathway. The Nedd4-like proteins contain WW substrate recognition domains, which recognise and bind proline–rich PY motifs. This work focuses on the interaction between Nedd4 and PMEPA1, a membrane protein showing altered expression in prostate cancer and a known Nedd4 substrate. PMEPA1 is recognised as important in several cancers, although its detailed function is not yet known; it is upregulated in prostate cancer and has been postulated to decrease cellular androgen receptor (AR) via a negative feedback loop involving Nedd4 in a ubiquitin-proteasome dependent process. Misregulation of the AR response to testosterone is associated with a more advanced form of prostatic cancer and poor patient prognosis, for which there are currently few treatment options available. PMEPA1 contains two well-documented canonical (PPxY) PY motifs both required for interaction with Nedd4. This work details the identification and characterisation of a third motif with a variant PY (vPY) sequence, QPTY. Our findings indicate that the loss of this vPY motif leads to a significant reduction in Nedd4-dependent ubiquitination in vitro. In addition, the nature of the amino acid residues surrounding the vPY motif also appears to play a role in the functionality of the site. Alongside these experiments, immunodetection of protein levels in HeLa and LNCaP cell lysates was used in conjunction with confocal microscopy to shed light on the interactions between PMEPA1, Nedd4 and AR in vitro and in vivo. A possible non-proteasomal role for ubiquitination in the PMEPA1-AR interaction, as opposed to the simple ubiquitin-proteasome mediated degradation previously proposed, is discussed in the light of this new data. This work has expanded previous knowledge of the specificity of the PY-WW interaction, as well as providing a basis for further investigation, and possibly clinical targeting, of the role of PMEPA1 and AR in prostate cancer.