Identification and Characterization of RPK118, a Novel Sphingosine Kinase-1-binding Protein

Abstract

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...

Whole-exome sequencing combined with functional genomics reveals novel candidate driver cancer genes in endometrial cancer

“…RPS6KC1 mutations have been previously found in breast, ovary, and lung cancers Stephens et al 2005;Cancer Genome Atlas Research Network 2011). Two independent studies show that RPS6KC1 does not have kinase activity (Hayashi et al 2002;Liu et al 2005), but, instead, likely functions as an adaptor molecule to recruit binding partners (sphingosine kinase-1 and peroxiredoxin-3) to early endosomes (Hayashi et al 2002;Liu et al 2005). These trafficking pathways within the endosomal system play a fundamental role in regulating protein degradation, recycling, secretion, and compartmentalization; and, indeed, defective vesicular trafficking is a hallmark of malignant transformation (Mosesson et al 2008).…”

Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

“…Subcellular localization studies using confocal microscopy were performed as described previously (11). A rabbit polyclonal anti-hSPHK2 antibody was raised against the synthetic peptide SQALHIQRLRPKPEARPR (amino acid residues 35-52) conjugated to keyhole limpet hemocyanin.…”

“…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.…”

Mouse Sphingosine Kinase Isoforms SPHK1a and SPHK1b Differ in Enzymatic Traits Including Stability, Localization, Modification, and Oligomerization

“…Sphingosine kinases are activated by stimuli such as treatment with platelet-derived growth factor (18), tumor necrosis factor ␣ (19), or phorbol ester (20) as well as the cross-linking of Fc␥R1 (21) or Fc⑀R1 (22). Additionally, SPHK1 is known to be regulated by protein-protein interactions (23)(24)(25)(26)(27), phosphorylation (28,29), and translocation to the plasma membrane (28,29). Although such regulation is involved in the stimuli-dependent activation of SPHK1, the precise molecular mechanisms that link the stimuli and the activation still remain largely unknown in most cases.…”