Julia R. Zebol

Activation of sphingosine kinase 1 by ERK1/2‐mediated phosphorylation

Sphingosine kinase 1 is an agonist‐activated signalling enzyme that catalyses the formation of sphingosine 1‐phosphate, a lipid second messenger that has been implicated in a number of agonist‐driven cellular responses, including stimulation of cell proliferation, inhibition of apoptosis and expression of inflammatory molecules. Although agonist‐induced stimulation of sphingosine kinase activity is critical in a number of signalling pathways, nothing has been known of the molecular mechanism of this activation. Here we show that this activation results directly from phosphorylation of sphingosine kinase 1 at Ser225, and present several lines of evidence to show compellingly that the activating kinase is ERK1/2 or a close relative. Furthermore, we show that phosphorylation of sphingosine kinase 1 at Ser225 results not only in an increase in enzyme activity, but is also necessary for translocation of the enzyme from the cytosol to the plasma membrane. Thus, these studies have elucidated the mechanism of agonist‐mediated sphingosine kinase activation, and represent a key finding in understanding the regulation of sphingosine kinase/sphingosine 1‐phosphate‐controlled signalling pathways.

Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca2+-dependent manner at the previously identified “calmodulin-binding site” of SK1. We also demonstrate that CIB1 functions as a Ca2+-myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling.

Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis

Sphingosine 1‐phosphate (S1P) is a bio‐active lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator‐activated receptor (PPAR) γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase‐tagged PPAR‐γ‐specific gene reporter by ~12‐fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPAR‐γ ligand binding domain is important for binding to S1P. PPAR‐γ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator‐activated receptor‐γ coactivator 1 (PGC1)β binds to PPAR‐γ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPAR‐γ:PGC1β complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPAR‐γ target genes with PGC1β and plasminogen‐activated inhibitor‐1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P‐induced in vitro tube formation was significantly attenuated in the presence of the PPAR‐γ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPAR‐γ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1‐/‐ Sphk2+/‐ mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPAR‐γ, is a bona fide intracellular target for S1P and thus suggest that the SlP:PPAR‐γ:PGC1β complex may be a useful target to manipulate neovascularization.—Parham, K. A., Zebol, J. R., Tooley, K. L., Sun, W. Y., Moldenhauer, L. M., Cockshell, M. P., Gliddon, B. L., Moretti, P. A., Tigyi, G., Pitson, S. M., Bonder, C. S. Sphingosine 1‐phosphate is a ligand for peroxisome proliferator‐activated receptor‐γ that regulates neoangiogenesis. FASEB J. 29, 3638‐3653 (2015). www.fasebj.org

The CCT/TRiC chaperonin is required for maturation of sphingosine kinase 1.

Sphingosine kinase 1 (SK1) catalyses the generation of sphingosine 1-phosphate (S1P), a bioactive phospholipid that influences a diverse range of cellular processes, including proliferation, survival, adhesion, migration, morphogenesis and differentiation. SK1 is controlled by various mechanisms, including transcriptional regulation, and post-translational activation by phosphorylation and protein-protein interactions which can regulate both the activity and localisation of this enzyme. To gain a better understanding of the regulatory mechanisms controlling SK1 activity and function we performed a yeast two-hybrid screen to identify SK1-interacting proteins. Using this approach we identified that SK1 interacts with subunit 7 (eta) of cytosolic chaperonin CCT (chaperonin containing t-complex polypeptide, also called TRiC for TCP-1 ring complex), a hexadecameric chaperonin that binds unfolded polypeptides and mediates their folding and release in an ATP-dependent manner. Further analysis of the SK1-CCTeta interaction demonstrated that other CCT/TRiC subunits also associated with SK1 in HEK293T cell lysates in an ATP-sensitive manner, suggesting that the intact, functional, multimeric CCT/TRiC complex associated with SK1. Furthermore, pulse-chase studies indicated that CCT/TRiC binds specifically to newly translated SK1. Finally, depletion of functional CCT/TRiC through the use of RNA interference in HeLa cells or temperature sensitive CCT yeast mutants reduced cellular SK1 activity. Thus, combined this data suggests that SK1 is a CCT/TRiC substrate, and that this chaperonin facilitates folding of newly translated SK1 into its mature active form.

An oncogenic role for sphingosine kinase 2

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an anti-proliferative/pro-apoptotic function for SK2, while others indicate it has a pro-survival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.

A point mutant of human sphingosine kinase 1 with increased catalytic activity

Sphingosine kinase (SK) catalyses the formation of sphingosine 1‐phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly113, a highly conserved residue of human sphingosine kinase 1 (hSK), by site‐directed mutagenesis. Surprisingly, a Gly113→Ala substitution generated a mutant that had 1.7‐fold greater catalytic activity than wild‐type hSK (hSKWT). Our data suggests that the Gly113→Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSKG113A activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSKWT.

Sphingosine kinase 2 inhibition synergises with bortezomib to target myeloma by enhancing endoplasmic reticulum stress

The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro. The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.

Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.