Growth factor mediated phosphatidylinositol 3 kinase/Akt/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) signalling pathway regulates a variety of cellular activities, including protein translation, cell metabolism and cell growth. PI3K/Akt signalling pathway is frequently dysregulated in prostate cancer due to inactivation of the tumour suppressor phosphatase and tensin homolog (PTEN), and is responsible for tumour formation and development. Their downstream effector mTORC1 can integrate cellular signals as well as nutrient levels, such as leucine, to coordinate cellular activities. Leucine is an essential amino acid that participates in protein synthesis and provides metabolic intermediates, and it also serves as an mTORC1 signalling regulator. In prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1). Therefore, we hypothesise that PI3K/Akt may regulate intracellular leucine concentration through LAT3. We have found that LAT3 is required for PI3K/Akt regulated leucine transport, and inhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines. With stimulation by EGF, leucine uptake increased significantly along with the activation of the PI3K/Akt signalling pathway. Knockdown of LAT3 effectively blocks leucine uptake, and this phenomenon cannot be rescued by growth factor addition or further inhibited by signalling pathway inhibition. Moreover, epidermal growth factor (EGF) significantly increases LAT3 protein levels when Akt is phosphorylated, and induces co-localisation of Akt and LAT3 on the plasma membrane in LNCaP cells. These effects are likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation.
We next set out to understand how LAT3 might be regulated by binding partners. To do this, we used a genetic code expansion method to incorporate unnatural amino acid (Uaa) into target proteins. This method has been widely used to study protein structure and dynamics in protein-protein interactions, as Uaas contain distinct chemical or physical properties compared to natural amino acids. We employed a photo-crosslinking Uaa, AzF, coupled with mass spectrometry to investigate the potential interactors of LAT3. We generated a series of LAT3 mutants, and successfully incorporated AzF into specific residues. With UV induction, we observed migration of protein bands on western blots, indicating the formation of protein complex by AzF contained LAT3. We further investigated this complex by immunoprecipitation or in-gel enzymatic digestion, coupled with mass spectrometry study. We have generated many candidate proteins by comparing different conditions and different sites. Although we haven’t been able to confirm particular interactor(s) of LAT3, this work has expanded our understanding of probing protein-protein interactions, and has laid the foundation for future investigations.
Finally, we set out to further understand the role of the essential amino acid leucine, one of the main LAT3 substrates, in cellular metabolism. Using radio-labelled leucine, we have observed the incorporation pattern of leucine under inhibition of PI3K/Akt signalling pathway or inhibition of transporter in prostate cancer cells. Leucine can be converted into glutamate and α-ketoglutarate through transamination, and participates in tricarboxylic acid (TCA) cycle, thus being incorporated into different cellular fractions. LNCaP cells exhibit decrease in most fractions under inhibition, however, PC-3 cells show increased incorporation into protein fraction under transporter inhibition.
This work has expanded our knowledge and understanding of LAT3, and its regulation by growth factor and PI3K/Akt signalling pathway in prostate cancer. The establishment of genetic code expansion method and mass spectrometry analysis provides new approaches to study membrane proteins like amino acid transporters and protein-protein interactions. In addition, the study of leucine metabolism in prostate cancer cells has provided insights into the utilisation of leucine and the adaptation of metabolism by prostate cancer cells. Future work will be needed to elucidate these aspects in more detail, and to identify vulnerability in metabolic pathway of leucine and to develop suitable therapeutic strategy targeting leucine metabolic pathway and/or LAT3 to improve prostate cancer treatment.