Information on EC 3.1.3.78 - phosphatidylinositol-4,5-bisphosphate 4-phosphatase

phosphatidylinositol-4,5-bisphosphate 4-phosphatase I

Q86T03

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phosphatidylinositol-4,5-bisphosphate 4-phosphatase II

Q8N4L2

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Salmonella enterica subsp. enterica serovar Typhimurium

phosphoinositide phosphatase

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PtdIns(4,5)P2 4-phosphatase

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715020

PtdIns-4,5-P2 4-phosphatase type I

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PtdIns-4,5-P2 4-phosphatase type II

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SigD

3 entries

type I 4-phosphatase

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type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase

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type I PtdIns-4,5-P2 4-Ptase

Q86T03

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type II 4-phosphatase

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type II PtdIns-4,5-P2 4-Ptase

Q8N4L2

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3-phosphoinositide degradation, D-myo-inositol-5-phosphate metabolism

1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate + H2O = 1-phosphatidyl-1D-myo-inositol 5-phosphate + phosphate

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MetaCyc

1-phosphatidyl-1D-myo-inositol-4,5-bisphosphate 4-phosphohydrolase

Two pathways exist in mammalian cells to degrade 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate [PtdIns(4,5)P2] [2]. One is catalysed by this enzyme and the other by EC 3.1.3.36, phosphoinositide 5-phosphatase, where the product is PtdIns4P. The enzyme from human is specific for PtdIns(4,5)P2 as substrate, as it cannot use PtdIns(3,4,5)P3, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns5P, PtdIns4P or PtdIns3P [2]. In humans, the enzyme is localized to late endosomal/lysosomal membranes [2]. It can control nuclear levels of PtdIns5P and thereby control p53-dependent apoptosis [3].

1D-myo-inositol 1,2,4,5,6-pentakisphosphate + H2O

1D-myo-inositol 1,4,5,6-tetrakisphosphate + phosphate

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?

D-myo-phosphatidylinositol 4,5-bisphosphate

D-myo-phosphatidylinositol 5-phosphate

-

-

?

inositol 1,3,4,5-tetrakisphosphate + H2O

inositol 1,3,5-trisphosphate + phosphate

-

-

?

inositol 1,4,5-trisphosphate + H2O

inositol 1,5-bisphosphate + phosphate

-

-

?

phosphatidylinositol 3,4,5-trisphosphate + H2O

phosphatidylinositol 3,5-bisphosphate + phosphate

3 entries

phosphatidylinositol 3,4-bisphosphate + H2O

phosphatidylinositol 3-phosphate + phosphate

-

-

?

phosphatidylinositol 3-phosphate + H2O

phosphatidylinositol + phosphate

-

Salmonella enterica subsp. enterica serovar Typhimurium

-

?

phosphatidylinositol 4,5-bisphosphate + H2O

phosphatidylinositol 5-monophosphate + phosphate

-

-

?

phosphatidylinositol 4,5-bisphosphate + H2O

phosphatidylinositol 5-phosphate + phosphate

-

-

?

phosphatidylinositol 4-phosphate + H2O

phosphatidylinositol + phosphate

-

-

?

phosphatidylinositol-4,5-bisphosphate + H2O

phosphatidylinositol-5-phosphate + phosphate

Salmonella enterica subsp. enterica serovar Typhimurium

additional information

?

-

3 entries

phosphatidylinositol 3,4,5-trisphosphate + H2O

phosphatidylinositol 3,5-bisphosphate + phosphate

O30916

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Salmonella enterica subsp. enterica serovar Typhimurium

-

?

phosphatidylinositol 4,5-bisphosphate + H2O

phosphatidylinositol 5-monophosphate + phosphate

-

-

?

phosphatidylinositol 4,5-bisphosphate + H2O

phosphatidylinositol 5-phosphate + phosphate

Q07566

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-

?

phosphatidylinositol-4,5-bisphosphate + H2O

phosphatidylinositol-5-phosphate + phosphate

Q86T03, Q8N4L2

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-

?

additional information

?

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PIPKIIbeta

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inhibits p53 acetylation and cell death

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RNAi

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siRNA of type I 4-phosphatase reduces type I 4-phosphatase levels, which is followed by a dramatic decrease in p53 levels in response to the genotoxic agent etoposide. siRNA of type II 4-phosphatase does not reduce p53 expression

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etoposide

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significantly increases type I 4-phosphatase in the nuclear fraction, in both the endogenous and overexpression system

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000003

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approx., C462A mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000005

-

approx., R468A mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000007

-

approx., GST, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000011

-

approx., DELTA1-351 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000015

-

approx., DELTA352-563 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000032

-

approx., DELTA118-142 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

Salmonella enterica subsp. enterica serovar Typhimurium

0.0000063

-

approx., SigD, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)

0.0003

10 microM concentration of inositol 1,4,5-trisphosphate and 1500 counts per minute of [3H]inositol 1,4,5-trisphosphate, recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg), reaction 37°C for 10 min in a 50 microl final volume containing 50 mM HEPES pH 7.4, 0.1% bovine serum albumin, 2 mM MgCl2, and 0.033% b-mercaptoethanol, similar results when expressed without IpgE

0.0058

50 microl of recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg) in 50 mM Tris pH 7.5 containing 50 mM glutathione and 100 mM NaCl , 37°C, 15 min, reaction mixture contained 10 microM [32P] phosphatidylinositol 3-phosphate and 20 microM phosphatidylserine, similar results when expressed without IpgE

0.01

50 microl of recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg) in 50 mM Tris pH 7.5 containing 50 mM glutathione and 100 mM NaCl , 37°C, 15 min, reaction mixture contained 10 microM [32P] phosphatidylinositol 4-phosphate and 20 microM phosphatidylserine, similar results when expressed without IpgE

0.0253

50 microl of recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg) in 50 mM Tris pH 7.5 containing 50 mM glutathione and 100 mM NaCl, 37°C, 15 min, reaction mixture contained 10 microM [32P] phosphatidylinositol 3,4-bisphosphate and 20 microM phosphatidylserine, similar results when expressed without IpgE

0.0321

50 microl of recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg) in 50 mM Tris pH 7.5 containing 50 mM glutathione and 100 mM NaCl, 37°C, 15 min, reaction mixture contained 10 microM [32P] phosphatidylinositol 3,4,5-trisphosphate and 20 microM phosphatidylserine, similar results when expressed without IpgE

0.061

50 microl of recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg) in 50 mM Tris pH 7.5 containing 50 mM glutathione and 100 mM NaCl , 37°C, 15 min, reaction mixture contained 10 microM [32P] phosphatidylinositol 4,5-bisphosphate and 20 microM phosphatidylserine, similar results when expressed without IpgE

additional information

Salmonella enterica subsp. enterica serovar Typhimurium

not detectable, 1 microM inositol 1,3,4,5-tetrakisphosphate and 1500 counts per minute of [3H]inositol 1,3,4,5-tetrakisphosphate, recombinant GST-IpgD expressed with chaperone IpgE (1-3 microg), reaction 37°C for 10 min in a 50 microl final volume containing 50 mM HEPES pH 7.4, 0.1% bovine serum albumin, 2 mM MgCl2, and 0.033% beta-mercaptoethanol

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SwissProt

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additional information

late; late

late; late

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when HeLa cells are treated with etoposide or doxorubicin, type I 4-phosphatase translocates to the nucleus and nuclear levels of phosphatidylinositol 5-phosphate increase

5634

physiological function

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PtdIns(4,5)P2 4-phosphatase expression induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T-cells. Expression of a PtdIns5P interacting domain, PH-Dok-5, blocks IpgD-induced T-cell activation and selective signaling molecules downstream of TCR triggering, evaluation, overview

715020

additional information

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phosphatidylinositol 5-phosphate may play a sensor function in setting the threshold of T-cell activation and contributing to maintain T-cell homeostasis

715020

C462S

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no functional or morphological changes in epithelial cells transfected with phosphatase-deficient mutant C462S

Salmonella enterica subsp. enterica serovar Typhimurium

C462A

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recombinant protein

Salmonella enterica subsp. enterica serovar Typhimurium

C462S

does not restore the ability of Salmonella to eliminate phosphatidylinositol 4,5-bisphosphate in SigD-deficient bacteria

Salmonella enterica subsp. enterica serovar Typhimurium

DELTA1-135-DELTA118-142

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recombinant protein, Saccharomyces cerevisiae: no effect in Saccharomyces cerevisiae, HeLa: no effect (Green Fluorescent Protein linked) in HeLa

Salmonella enterica subsp. enterica serovar Typhimurium

DELTA1-165

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recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments

Salmonella enterica subsp. enterica serovar Typhimurium

DELTA1-351

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recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments

Salmonella enterica subsp. enterica serovar Typhimurium

DELTA118-142

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recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, residues 118–142 crucial for toxicity of SigD in the yeast cells, affecting both phosphatase activity and actin depolarization events, no effect (Green Fluorescent Protein linked) in HeLa

Salmonella enterica subsp. enterica serovar Typhimurium

DELTA352-563

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recombinant protein, no effect in Saccharomyces cerevisiae, no effect in HeLa (Green Fluorescent Protein linked)

Salmonella enterica subsp. enterica serovar Typhimurium

K527A

restores the ability of Salmonella to eliminate phosphatidylinositol 4,5-bisphosphate in SigD-deficient bacteria

Salmonella enterica subsp. enterica serovar Typhimurium

K530A

restores the ability of Salmonella to eliminate phosphatidylinositol 4,5-bisphosphate in SigD-deficient bacteria

Salmonella enterica subsp. enterica serovar Typhimurium

R468A

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recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments

C438S

site-directed mutagenesis

additional information

FLAG-tagged isozyme I from Sf9 insect cells by anti-FLAG immunoaffinity chromatography; FLAG-tagged isozyme II from Sf9 insect cells by anti-FLAG immunoaffinity chromatography

Q86T03, Q8N4L2

Salmonella enterica subsp. enterica serovar Typhimurium

(PCR) amplification from the SigD open reading frame from S. enterica ser. typhimurium C53 genomic DNA. 2 types plasmids: first type based on the pEG-KG vector to express GST fusion proteins in yeast, second type based on pGEX-KG for expression of GST-fused proteins in bacteria. transformation of Escherichia coli DH5alpha cells (used for molceularbiological methods and expression). Transformation of Saccharomyces cerevisiae YPH499 (used for SigD functional analyzes as a model organism). transfection of human epithelial cell line HeLa (ATCC CCL2). yeast: endogenous SigD expression results in severe growth inhibition, allele mutant in catalytic site or deletion of whole C-terminal phosphatase domain inhibit growth by loss of actin polarization and precluding the budding process. HeLa: expression of same sigD alleles causes loss of ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, actin fibres still disappear and lose their adherence. region of 25 amino acids localized (residues 118–142) essentially required for the effect of SigD on actin in HeLa cells. SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity

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by cloning SigD into a mammalian expression vector, it is introduced into intact epithelia cells by microinjection: wild-type SigD induces striking morphological and functional changes that are not mimicked by a phosphatase-deficient SigD mutant C462S

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infection assay: isotopic equilibrium of Shigella flexneri either wild-type strain M90T, ipgD mutant or the non-invasive strain BS176 (each expressing the AfaE adhesion). Green Fluorescent Protein-tagged IpgD recombinant protein (2 types: wild-type and C438S) cloned in pKN16, transfection of semi-confluent HeLa cells or NIH-3T3 cells. IpgD hydrolyses phosphatidylinositol 4,5-bisphosphate during infection of epithelial cells by Shigella flexneri (analysis of phospholipid content of cells labelled with 32P), phosphatidylinositol 5-phosphate is the product of IpgD-dependent phosphatidylinositol 4,5-bisphosphate degradation in Shigella flexneri infected cells (HPLC, mass assay, immunofluorescence), cellular phosphatidylinositol 4,5-bisphosphate is the only phosphoinositide hydrolysed in vivo, IpgD causes membrane blebbing and cell rounding when expressed in HeLa cells (transiently expressed myc-tagged IpgD in HeLa cells, SDS-PAGE, antibodies against tag or protein), IpgD decreases cytoskeletal-membrane adhesion when expressed in NIH-3T3 cells (optical tweezers)

Salmonella enterica subsp. enterica serovar Typhimurium

Salmonella typhimurium SL1344 wild-type and deltaSigD mutant lacking SigD. Transfection of COS-7 and HeLa cells with SigD in mammalian expression vectors. Visualization of phosphatidylinositol 4,5-bisphosphate during Salmonella infection. Fusion protein of PH domain of phospholipase Cdelta and GFP (PLCdelta-PH-GFP) during invasion of HeLA cells by Salmonella (confocal fluorescence microscopy): phosphatidylinositol 4,5-bisphosphate is abundant near the tip but disappears from the basal regions. Role of SigD in phosphatidylinositol 4,5-bisphosphate elimination: focal disappearance of phosphatidylinositol 4,5-bisphosphate observed during invasion (wild-type) is diminished and delayed in the deltaSigD mutant (confocal fluorescence microscopy). SigD alters memebrane elasticity (atomic force microscopy, SigD transfected HeLa cells) and induces vacuole formation. SigD promotes membrane fission during invasion (SigD transfected HeLa cells)

stably transfected HEK-293 TRex cells in a tetracycline-inducible vector expressing type I and II 4-phosphatase

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type II isozyme, DNA and amino acid sequence determination and analysis, transient transfection of functional enzyme to HeLa and COS-7 cells leading to increased EGFR degradation via increased phosphatidylinositol-5-phosphate content, functional overexpression of FLAG-tagged enzyme in Spodoptera frugiperda Sf9 cells using the baculovirus infection system; type I isozyme, DNA and amino acid sequence determination and analysis, transient transfection of functional enzyme to HeLa and COS-7 cells leading to increased EGFR degradation via increased phosphatidylinositol-5-phosphate content, functional overexpression of FLAG-tagged enzyme in Spodoptera frugiperda Sf9 cells using the baculovirus infection system, stable inducible expression of labeled isozyme I in HEK293 cells

Q86T03, Q8N4L2