Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase.
Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.
Ross
T S
TS
Washington University School of Medicine, Division of Hematology-Oncology, St. Louis, Missouri 63110.
Jefferson
A B
AB
Mitchell
C A
CA
Majerus
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D010957:Plasmids; D011233:Precipitin Tests; D012313:RNA / Q000032:analysis; D011817:Rabbits
D015342:DNA Probes; D012313:RNA; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases