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 kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell. Sphingosine kinase (SPHK)1 is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events including cell growth, survival, motility, cytoskeletal changes, and the release of calcium from intracellular stores (1, 2) by acting both as an extracellular agonist and an intracellular messenger (3). The extracellular effects of SPP are mediated by the recently identified endothelial differentiation gene (EDG) receptors, novel members of the G-proteincoupled heptahelical receptor family (4). For example, the binding of SPP to HEK293 cells stably expressing EDG-1 induced the inhibition of cAMP accumulation and the activation of extracellular signal-regulated kinase (ERK) in a pertussis toxin-sensitive manner (5). On the other hand, the following findings were important clues to a specific intracellular action of SPP. First, the activation of various plasma membrane receptors such as the platelet-derived growth factor (6, 7) and the Fc⑀RI (8) was found to rapidly increase intracellular SPP production through the stimulation of SPHK. Second, microinjected SPP mobilized Ca 2ϩ from the internal stores of cells that had been pretreated with pertussis toxin to inactivate G i -or G o -coupled receptor signaling (9). Third, the manipulation of intracellular SPP content in yeast cells, which lack a cell surface receptor for SPP, by the overexpression or deletion of genes that encode SPHK has revealed an important role for SPP in yeast survival and proliferation during exposure to heat or nutrient-deprivation stress. However, the intracellular site of action of SPP remains unknown.The present studies were designed to determine the intracellular site of action of SPP by identifying molecules interacting with sphingosine kinase-1 (SPHK1) using a yeast...

We have previously reported that a heat-stable activator for ganglioside metabolism, GM2 activator, potently stimulates ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) activity (presumably PLD1) in an in vitro system [Nakamura, Akisue, Jinnai, Hitomi, Sarkar, Miwa, Okada, Yoshida, Kuroda, Kikkawa and Nishizuka (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12249–12253]. However, little is known about the regulation of PLD2. In the present studies we have investigated the regulation of PLD2 by GM2 activator and various other regulators including ARF. PLD2 was potently stimulated in vitro by GM2 activator in a time- and dose-dependent manner. Neither ARF nor protein kinase C caused any significant changes in PLD2 activity. Importantly, PLD2 responsiveness to ARF was greatly enhanced by GM2 activator, suggesting a possible role for GM2 activator as a coupling factor. GM2 activator was also demonstrated to physically associate with PLD2 in a stoichiometric manner. Further, PMA stimulation of COS-7 cells overexpressing both GM2 activator and PLD2 resulted in a marked increase in the association of the two molecules. Interestingly, ARF association with PLD2 was greatly increased by GM2 activator. Moreover, GM2 activator enhanced PMA-induced PLD activity in a synergistic manner with ARF in streptolysin-O-permeabilized, cytosol-depleted HL-60 cells, suggesting that GM2 activator may regulate PLD in a concerted manner with other factors, including ARF, inside the cells.

We have previously reported that a heat-stable activator for ganglioside metabolism, G M# activator, potently stimulates ADPribosylation factor (ARF)-dependent phospholipase D (PLD) activity (presumably PLD1) in an in itro system [Nakamura,