Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein.
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.
Mesh Terms:
Amino Acid Sequence, Animals, Binding Sites, Blotting, Northern, COS Cells, Carrier Proteins, Cloning, Molecular, DNA, Complementary, Genetic Vectors, Humans, Immunoblotting, Intracellular Signaling Peptides and Proteins, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, Phosphatidylinositol Phosphates, Phosphotransferases (Alcohol Group Acceptor), Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Rats, Ribosomal Protein S6 Kinases, Sequence Homology, Amino Acid, Signal Transduction, Tissue Distribution, Transfection, Two-Hybrid System Techniques
Amino Acid Sequence, Animals, Binding Sites, Blotting, Northern, COS Cells, Carrier Proteins, Cloning, Molecular, DNA, Complementary, Genetic Vectors, Humans, Immunoblotting, Intracellular Signaling Peptides and Proteins, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, Phosphatidylinositol Phosphates, Phosphotransferases (Alcohol Group Acceptor), Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Rats, Ribosomal Protein S6 Kinases, Sequence Homology, Amino Acid, Signal Transduction, Tissue Distribution, Transfection, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Sep. 06, 2002
PubMed ID: 12077123
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