Novel inositol polyphosphate 5-phosphatase localizes at membrane ruffles.
We have cloned a novel inositol polyphosphate 5-phosphatase from the rat brain cDNA library. It contains two highly conserved 5-phosphatase motifs, both of which are essential for its enzymatic activity. Interestingly, the proline content of this protein is high and concentrated in its N- and C-terminal regions. One putative SH3-binding motif and six 14-3-3 zeta-binding motifs were found in the amino acid sequence. This enzyme hydrolyzed phosphate at the D-5 position of inositol 1,4,5-trisphosphate, inositol 1,3,4, 5-tetrakisphosphate, and phosphatidylinositol 4,5-bisphosphate, consistent with the substrate specificity of type II 5-phosphatase, OCRL, synaptojanin and synaptojanin 2, already characterized 5-phosphatases. When the Myc-epitope-tagged enzyme was expressed in COS-7 cells and stained with anti-Myc polyclonal antibody, a signal was observed at ruffling membranes and in the cytoplasm. We prepared several deletion mutants and demonstrated that the 123 N-terminal amino acids (311-433) and a C-terminal proline-rich region containing 277 amino acids (725-1001) were essential for its localization to ruffling membranes. This enzyme might regulate the level of inositol and phosphatidylinositol polyphosphates at membrane ruffles.
Mochizuki
Y
Y
Department of Biochemistry, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Takenawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D000072181:Inositol Polyphosphate 5-Phosphatases; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases