Properties of Type II Inositol Polyphosphate 5-Phosphatase

doi:10.1074/jbc.270.16.9370

Properties of Type II Inositol Polyphosphate 5-Phosphatase (*)

From the (1) From Washington University School of Medicine, St. Louis, Missouri 63110

Abstract

We have isolated additional cDNA clones encoding type II inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a combined cDNA of 3076 nucleotides encoding a protein of 942 amino acids. The 5-phosphatase II hydrolyzed both Ins (1, 4, 5) P₃to Ins (1, 4) P₂and the phospholipid PtdIns (4, 5) P₂to PtdIns (4) P both in vitro and in vivo. There are two motifs highly conserved between types I and II 5-phosphatase and several other proteins presumed to be inositol phosphatases suggesting a possible role in catalysis. The type II 5-phosphatase also contains homology to several GTPase activating proteins although no such activity for 5-phosphatase II was found. The predicted protein ends with the sequence CNPL, suggesting that it is isoprenylated as a mechanism for membrane attachment. We found evidence for isoprenylation by demonstrating incorporation of [³H]mevalonate into native but not C939S mutant 5-phosphatase II expressed in Sf9 insect cells. Furthermore, we showed that membrane localization and the activity of 5-phosphatase II toward its lipid substrate PtdIns (4, 5) P₂is reduced by eliminating 5-phosphatase II isoprenylation in the mutant C939S relative to the native enzyme.

Footnotes

↵* This research was supported by National Institutes of Health Grants HL 14147 (Specialized Center for Research in Thrombosis), HL 16634, and Training Grant HL 07088. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PtdIns(4,5)P₂

phosphatidylinositol 4,5-bisphosphate

Ins(1,4,5)P₃

inositol 1,4,5-trisphosphate

Ins(1,3,4,5)P₄

inositol 1,3,4,5-tetrakisphosphate

Ins(1,4)P₂

inositol 1,4-bisphosphate

Ins(1,3,4)P₃

inositol 1,3,4-trisphosphate

GST

glutathione S-transferase

PCR

polymerase chain reaction

Mes

4-morpholineethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

GTPS

guanosine 5′- O-(3-thiotriphosphate

GAP

GTPase activating protein.
↵²P. Casey, personal communication.