Identification and Characterization of a Novel Inositol Polyphosphate 5-Phosphatase

Abstract

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4,5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K m ‫؍‬ 180 M) than for inositol 1,4,5-trisphosphate (K m ‫؍‬ 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 is known to play a role in the generation of two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) and diacylglycerol (DG), by phospholipase C. Ins(1,4,5)P 3 mobilizes Ca 2ϩ stores, and DG activates protein kinase C (1, 2).In addition to its role as a lipid second messenger, PtdIns(4,5)P 2 binds to several actin-regulating proteins such as vinculin, profilin, cofilin, and gelsolin (3-6) to suppress their functions. In contrast, PtdIns(4,5)P 2 binds also to ␣-actinin to activate the actin-gelating activity (7). An inositol polyphosphate 5-phosphatase (IP5Pase), synaptojanin 1, previously demonstrated to hydrolyze PtdIns(4,5)P 2 , bound to vinculin, ␣-actinin, and profilin in vitro (8). However, the molecular mechanism by which IP5Pase regulates the actin cytoskeleton remains to be elucidated.IP5Pases hydrolyze the D-5 position of inositol phosphates and corresponding phospholipids. Eight IP5Pases and several splicing variants were isolated and classified into three groups according to their substrate specificity (9 -24). Type I 5-phosphatases hydrolyze only water-soluble substrates, Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 , indicating roles in regulating intracellular calcium levels (11). Type II enzymes hydrolyze PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 as well as soluble inositol phosphates. OCRL encodes the X chromosome, and its mutation causes Lowe's oculocerebrorenal syndrome (10). Synaptojanin 1 and 2 are related enzymes (14,19,20). They contain C-terminal proline-rich regions that bind SH3 homology domains of Ash/Grb2 or amphiphysin and an N-terminal domain homologous to yeast protein SacI, recently identified as inositol polyphosphate phosphatase (24). PIPP (Proline-rich Inositol Polyphosphate 5-Phosphatase) is a recently identified Type II IP5Pase that localizes at membrane ruffles (25). Type III...

Identification of a Novel Domain in Two Mammalian Inositol-polyphosphate 5-Phosphatases That Mediates Membrane Ruffle Localization

“…Kinetic analysis using recombinant 5-phosphatase indicates that the enzyme's major substrates in vitro are PtdIns(3,4,5)P 3 and PtdIns(4,5)P 2 . Ectopic overexpression of SKIP in COS-7 cells results in loss of actin stress fibers in the regions of overexpression, presumably via regulation of PtdIns(4,5)P 2 and thereby the actin cytoskeleton (19). PIPP is a 107-kDa 5-phosphatase that hydrolyzes PtdIns(4,5)P 2 , Ins(1,4,5)P 3 , and Ins(1,3,4,5)P 4 and localizes to membrane ruffles (20).…”

“…Two novel mammalian 5-phosphatases designated SKIP for skeletal muscle and kidney enriched inositol phosphatase (19) and PIPP for proline-rich inositol-polyphosphate 5-phospha-tase (20) have recently been cloned, but not extensively characterized. The 5-phosphatase SKIP is a 51-kDa enzyme that contains a central catalytic 5-phosphatase domain with no other reported motifs.…”

“…Humans deficient in OCRL 5-phosphatase (16) demonstrate growth and mental retardation, renal tubular acidosis, and cataracts, although mice that lack this enzyme surprisingly show no phenotype (17). Homozygous male 5-phosphatase II-deficient mice are infertile due to disrupted sperm function (18).Two novel mammalian 5-phosphatases designated SKIP for skeletal muscle and kidney enriched inositol phosphatase (19) …”

“…Two novel mammalian 5-phosphatases designated SKIP for skeletal muscle and kidney enriched inositol phosphatase (19) …”

Mutations in INPP5K Cause a Form of Congenital Muscular Dystrophy Overlapping Marinesco-Sjögren Syndrome and Dystroglycanopathy

“…Also known as skeletal muscle and kidney-enriched inositol phosphatase (SKIP), INPP5K is highly expressed in the developing and adult brain, eye, and muscle. [12][13][14] It is primarily localized to the ER, and it can form a complex with HSPA5/BiP to regulate insulin receptor signaling at actin ruffles on the plasma membrane by acting as a negative regulator of phosphatidylinositol-3-ki- Table 1 for numeric values.…”