Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

Abstract

Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G 1 /S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.Sphingosine 1-phosphate (SPP) 1 is a bioactive lipid that regulates diverse biological processes such as calcium mobilization, cell growth, differentiation, survival, motility, and cytoskeletal reorganization, acting both inside and outside the cells (1, 2). Recently, SPP was identified as the ligand for a family of G protein-coupled receptors known as the endothelial differentiation gene-1 family, now collectively renamed SPP receptors (3-6), supporting a role for SPP as an extracellular ligand. However, the intracellular targets of SPP have not yet been identified.Sphingosine kinase (SPHK), the enzyme that catalyzes the phosphorylation of sphingosine, regulates the intracellular levels of SPP. Two isoforms of mammalian SPHK (SPHK1 and SPHK2) have been cloned and characterized (7,8). SPHK1 predominantly localizes in the cytosol, and its overexpression induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (9). Several cellular proteins have recently been identified as SPHK1-interacting molecules, namely TRAF2 (10), RPK118 (11), and AKAP-related protein (12), which should help facilitate the understanding of the regulation and intracellular site of action of SPHK1.In contrast to SPHK1, little is known about the cellular actions of the other isozyme, SPHK2. In the present studies, we investigated the biological features of SPHK2. We have discovered that SPHK2 localizes in the nuclei of cells through its novel nuclear localization signal (NLS) sequence, depending on cell type and cell density. We have also demonstrated that nuclear localization of SPHK2 causes inhibition of DNA synthesis in various cell types.

Sphingosine-1-phosphate promotes expansion of cancer stem cells via S1PR3 by a ligand-independent Notch activation

“…We next examined whether SphK is involved in breast CSCs. Consistent with previous reports 32,33 , overexpressed SphK1 was localized in the cytosol, and SphK2 was mainly localized to the nucleus (Fig. 5a).…”

Subcellular distribution of FTY720 and FTY720-phosphate in immune cells – another aspect of Fingolimod action relevant for therapeutic application

“…We distinguished four fractions: (1) medium surrounding the cells, (2) cytosol, (3) nucleus and (4) the membranous compartments. Because the major FTY720 bio-activating enzyme SphK2 is located primarily in the nuclear and membranous compartments (Igarashi et al, 2003;Hait et al, 2009) we were interested to analyze how FTY720 and FTY720-P are distributed not only in wild type mice, but also in the splenocytes deficient for SphK2. Furthermore, our concern was to determine whether the distribution of FTY720 and FTY720-P is affected in the absence of the enzyme SphK1, phosphorylating S1P extranuclearly.…”

SphK1 and SphK2, Sphingosine Kinase Isoenzymes with Opposing Functions in Sphingolipid Metabolism

“…In stark contrast, overexpression of SphK2 does the opposite, suppressing cell growth and inducing apoptosis (26,27). Previously we have shown that mutation of leucine 219 in its putative BH3 domain, a highly conserved leucine present in all BH3 domains (28), reduced its ability to induce apoptosis (26) (Fig.…”

“…These two isoenzymes have different kinetic properties and also differ in developmental and tissue expression (16), implying that they may have distinct physiological functions. Indeed, rather than promoting growth and survival, SphK2 suppressed growth and enhanced apoptosis that was preceded by cytochrome c release and activation of caspase-3 (26,27). Moreover, SphK2-induced apoptosis was independent of activation of S1P receptors (26).…”