Cloning, and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network

被引:79
作者
Kong, AM
Speed, CJ
O'Malley, CJ
Layton, MJ
Meehan, T
Loveland, KL
Cheema, S
Ooms, LM
Mitchell, CA [1 ]
机构
[1] Monash Univ, Dept Biochem & Mol Biol, Clayton, Vic 3168, Australia
[2] Monash Univ, Dept Anat & Cell Biol, Clayton, Vic 3168, Australia
[3] Monash Med Ctr, Monash Inst Reprod & Dev, Clayton, Vic 3168, Australia
[4] Walter & Eliza Hall Inst Med Res, Parkville, Vic 3050, Australia
[5] Ludwig Inst Canc Res, Joint Prot Struct Lab, Parkville, Vic 3050, Australia
关键词
D O I
10.1074/jbc.M000874200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The inositol-polyphosphate B-phosphatase enzyme family removes the B-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel B-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central B-phosphatase domain, and a C-terminal CAAX motif. The 3.9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa B-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol S-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.
引用
收藏
页码:24052 / 24064
页数:13
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