Stuart M. Pitson

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.

An oncogenic role of sphingosine kinase

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Regulation of sphingosine kinase and sphingolipid signaling

Bioactive sphingolipids, including ceramide, sphingosine and sphingosine 1-phosphate are important regulators of many cellular processes, including cell survival, proliferation, differentiation, migration and immune responses. Although the levels of these bioactive sphingolipids are regulated by complex pathways subject to spatial and temporal control, the sphingosine kinases have emerged as critical central regulators of this system and, as a consequence, they have received substantial recent attention as potential therapeutic targets for cancer and a range of other conditions. Deciphering the molecular mechanisms that regulate both the activity and subcellular localization of these enzymes is vital for understanding the control of bioactive sphingolipid generation and action, and has clear implications for therapeutic strategies targeting these enzymes.

Expression of a Catalytically Inactive Sphingosine Kinase Mutant Blocks Agonist-induced Sphingosine Kinase Activation A DOMINANT-NEGATIVE SPHINGOSINE KINASE

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly82 to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-α (TNFα), interleukin-1β, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSKG82D). Basal SK activity was unaffected by expression of hSKG82D. Expression of hSKG82D had no effect on TNFα-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSKG82D acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFα activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

Essential Roles of Sphingosine‐1‐Phosphate and Platelet‐Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum—sphingosine‐1‐phosphate (S1P), lysophosphatidic acid (LPA), and platelet‐derived growth factor (PDGF)—in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum‐free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri‐potentiality.

Noncellulolytic fungal β-glucanases: Their physiology and regulation

The occurrence, regulation, and action of fungal enzymes capable of degrading noncellulosic β-glucans, especially 1,3-β- and 1,6-β-glucans, are reviewed. Special consideration is given to their roles in both metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, and autolysis. Also examined are the protocols currently available for their purification, with some of the properties of purified β-glucanases discussed in terms of their potential applications in industrial, agricultural, and medical fields.

Sphingosine Kinase Modulates Microvascular Tone and Myogenic Responses Through Activation of RhoA/Rho Kinase

Background RhoA and Rho kinase are important modulators of microvascular tone. Methods and Results We tested whether sphingosine kinase (Sphk1) that generates the endogenous sphingolipid mediator sphingosine‐1‐phosphate (S1P) is part of a signaling cascade to activate the RhoA/Rho kinase pathway. Using a new transfection model, we report that resting tone and myogenic responses of isolated resistance arteries increased with forced expression of Sphk1 in smooth muscle cells of these arteries. Overexpression of a dominant negative Sphk1 mutant or coexpression of dominant negative mutants of RhoA or Rho kinase together with Sphk1 completely inhibited development of tone and myogenic responses. Conclusions The tone‐increasing effects of a Sphk1 overexpression suggest that Sphk1 may play an important role in the control of peripheral resistance. (Circulation. 2003;108:342‐347.)

FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod™) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called ‘inside-out’ signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P2/3 receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P2/3 receptors.

The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function

The sphingosine and diacylglycerol kinases form a superfamily of structurally related lipid signaling kinases. One of the striking features of these kinases is that although they are clearly involved in agonist-mediated signaling, this signaling is accomplished with only a moderate (and sometimes no) increase in the enzymatic activity of the enzymes. Here, we summarize findings that indicate that signaling by these kinases is strongly dependent on their localization to specific intracellular sites rather than on increases in enzyme activity. Both the substrates and products of these enzymes are bioactive lipids. Moreover, many of the metabolic enzymes that act on these lipids are found in specific organelles. Therefore, changes in the membrane localization of these signaling kinases have profound effects not only on the production of signaling lipid phosphates but also on the metabolism of the upstream signaling lipids.

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.