Sphingosine Kinase 1 Isoform-Specific Interactions in Breast Cancer

Abstract

Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor β-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

“…Numerous commercial anti-SphK1 & anti-phospho SphK1 antibodies were screened to identify antibodies for detection and immunoprecipitation of SphK1 protein from lysates obtained of TGFβ1-treated monolayer cell cultures. Several groups have described multiple SphK1 protein isoforms via Western blotting (26). We report similar results in our studies where 2-3 predominant isoforms (between 41-53kDa) can be detected by Western blotting in a time-dependent manner upon treatment with either TGFβ1 or Activin A with peak protein accumulation occurring at 24hrs (Fig.…”

Annexin/S100A Protein Family Regulation through p14ARF-p53 Activation: A Role in Cell Survival and Predicting Treatment Outcomes in Breast Cancer

“…The method for SILAC metabolic triple labeling has been described previously [35]. Labeled amino acids, dialyzed fetal bovine serum (FBS), and lysine and arginine-free media (SILAC media) were purchased from Silantes GmbH.…”

“…MCF-7 cells were split and triple-labeled with three differentially labeled (‘light’, ‘medium’, and ‘heavy’) media formulations as previously described [35]. Proteins were metabolically labeled for a minimum of 6 doublings in lysine- and arginine-free DMEM medium containing 10% ( v / v ) dialyzed FBS and supplemented with either: (a) ‘light’, unlabeled lysine and arginine; (b) ‘medium’ lysine-4 ( 2 H 4 -lysine) and arginine-6 ( 13 C 6 -L-arginine); or (c) ‘heavy’ lysine-8 ( 13 C 6 15 N 2 -L-lysine) and arginine-10 ( 13 C 6 15 N 4 -L-arginine).…”

“…Lysates were fractionated into nuclear and cytoplasmic fractions for better peptide coverage. Cell lysates were separated by electrophoresis and in-gel tryptic digestion was carried out prior to analysis by high-resolution mass spectrometry [35]. Treated cells were harvested, counted and equal numbers of cells were combined from the different SILAC labeled cells in a 1:1:1 ratio and protein extracted.…”

“…Treated cells were harvested, counted and equal numbers of cells were combined from the different SILAC labeled cells in a 1:1:1 ratio and protein extracted. The extracted protein underwent quantitative proteomic analysis by tryptic digestion followed by tandem mass spectrometry (LC MS/MS) [35]. …”