Thesis
The role of a monomer/dimer equilibrium and the C-terminal tail in regulating translocation of sphingosine kinase 1 to the plasma membrane of breast cancer cells
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2020
- Thesis identifier
- T15787
- Person Identifier (Local)
- 201778512
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Sphingosine kinase-1 (SK1) is able to translocate from the cytoplasm to the plasma membrane (PM) to catalyse the formation of sphingosine 1-phosphate (S1P) from sphingosine and adenosine triphosphate (ATP). It is known that both the expression and activity of SK1 is increased in many tumour types leading to a poorer prognostic outcome in terms of disease-specific survival (Heffernan-Stroud et al., 2013). In addition, the translocation/activation of SK1 to the plasma membrane induces oncogenesis. However, the mechanism of translocation requires further investigation in order to identify new targets for potential therapeutic intervention in the treatment of cancer. Therefore, the aim of the current study was to further examine the molecular mechanisms regulating the translocation of SK1 from the cytoplasm to the PM in MCF-7L breast cancer cells. Phorbol myristate acetate (PMA) induced the extracellular signal-regulated kinase (ERK)-dependent translocation of wild type (WT) mouse green fluorescent protein (GFP)-SK1 (mGFP-SK1) from the cytoplasm to lamellipodia in the PM of MCF-7L breast cancer cells. In addition, carbachol also induced translocation of WTmGFP-SK1 to lamellipodia, albeit in an ERK-independent manner.;In contrast, S1P induced the ERK-independent translocation of WTmGFP-SK1 to filopodia. Treatment of cells with either the phospholipase D (PLD) inhibitor, 5-Fluoro-2-Indolyl des-chlorohalopemide (FIPI), or the Gq inhibitor, YM254890 reduced translocation in response to phorbol myristate acetate (PMA), carbachol or S1P, suggesting that phosphatidic acid (PA) formation and Gq activation are required for phosphorylation-dependent and -independent translocation of SK1 to the PM of breast cancer cells. It has previously been proposed that SK1 might be able to exist as monomer and dimer in equilibrium. The dimeric structure would allow for a contiguous membrane engagement interface consisting of hydrophobic patches on the lipid binding loop (LBL)-1 loop and a cluster of positively charged residues at the dimeric interface. The possibility of a monomer/dimer equilibrium was investigated by generating a constitutively monomeric mGFP-SK1 mutant, mGFP-SK1-K49E by introducing charge opposition in dimer interface.;We also created a stabilized dimer mGFP-SK1 mutant, mGFP-SK1-I51C by engineering a disulphide bond in dimer interface. Carbachol and PMA promotedt he translocation of mGFP-SK1-K49E to lamellipodia, whereas in contrast to WTmGFPSK1, S1P also induced translocation of mGFP-SK1-K49E to lamellipodia. On the other hand, carbachol or PMA promoted the translocation of mGFP-SK1-I51C to filopodia in a manner similar to S1P. These novel findings suggest monomeric and dimeric SK1 translocate to different PM micro-domains and that the position of the monomer/dimer equilibrium is determined by the ligand. We additionally investigated the regulatory role of the C-terminal tail of SK1 by creating mutants of mGFP-SK1, termed T1-T5 in which 5 amino acids were sequentially truncated with the exception of T4 in which 19 amino acids in total were removed. We identified that mGFP-SK1 T1 in which 5 amino acids were removed exhibited markedly reduced translocation in response to carbachol and S1P.;However, further truncation of the C-terminus restored the ability of SK1 to translocate to the PM. These results are consistent with molecular modelling studies in which displacement of the C-terminus 'locking motif' (C-terminal amino acids 6-10) might be achieved by the binding of a putative adapter protein to the 'displacement motif' (C-terminal amino acids 1-5). It is proposed that this displacement of the C-terminal tail enables NTD:CTD twisting around a 'connecting rod' to produce alignment of membrane engagement determinants; namely the positive charge cluster and LBL-1 to promote translocation of SK1. Moreover, molecular modelling studies also suggest that the ERK-catalysed Ser225 phosphorylation might destabilize the auto-inhibitory C-terminal folding independently of the binding of the adapter protein. These findings represent a unified mechanism of SK1 translocation to the PM, involving either ERK-mediated phosphorylation of Ser225 or displacement of the C-terminal tail by a putative adaptor protein. These novel findings provide new information concerning the mechanism of SK1 translocation that might assist in the identification of novel therapeutics aimed at perturbing translocation of this enzyme in cancer and to therefore reduce its oncogenic potential.
- Advisor / supervisor
- Pyne, Susan.
- Pyne, Nigel.
- Resource Type
- DOI
- Date Created
- 2020
- Former identifier
- 9912936793502996
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