Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: A comparison of PH-domain-mediated methods with immunological methods

Shu Chin Yip, Robert J. Eddy, Angie M. Branch, Huan Pang, Haiyan Wu, Ying Yan, Beth E. Drees, Paul O. Neilsen, John S. Condeelis, Jonathan M. Backer

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P 3 production using a specific monoclonal anti-PtdIns(3,4,5)P 3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3 production, measured by the membrane translocation of an epitope-tagged BTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3 hydrolysis by measuring the decay of the PtdIns(3,4,5)P3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P 3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3 in vitro. These data suggest that anti-PtdIns(3,4,5)P3 antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

Original languageEnglish (US)
Pages (from-to)441-448
Number of pages8
JournalBiochemical Journal
Volume411
Issue number2
DOIs
StatePublished - Apr 15 2008

Fingerprint

Epidermal Growth Factor
Cells
Carcinoma
Phosphatidylinositols
Membranes
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
phosphatidylinositol 3,4,5-triphosphate
Pleckstrin Homology Domains
platelet protein P47
PTEN Phosphohydrolase
Staining and Labeling
Chromosomes, Human, Pair 10
Kinetics
1-Phosphatidylinositol 4-Kinase
Antibodies
Cell membranes
Chromosomes
Green Fluorescent Proteins
Epitopes
Hydrolysis

Keywords

  • Carcinoma cell
  • Epidermal growth factor (EGF)
  • Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P]
  • Phosphoinositide
  • Phosphoinositide 3-kinase (PI3K)
  • Pleckstrin homology domain (PH domain)

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells : A comparison of PH-domain-mediated methods with immunological methods. / Yip, Shu Chin; Eddy, Robert J.; Branch, Angie M.; Pang, Huan; Wu, Haiyan; Yan, Ying; Drees, Beth E.; Neilsen, Paul O.; Condeelis, John S.; Backer, Jonathan M.

In: Biochemical Journal, Vol. 411, No. 2, 15.04.2008, p. 441-448.

Research output: Contribution to journalArticle

Yip, Shu Chin ; Eddy, Robert J. ; Branch, Angie M. ; Pang, Huan ; Wu, Haiyan ; Yan, Ying ; Drees, Beth E. ; Neilsen, Paul O. ; Condeelis, John S. ; Backer, Jonathan M. / Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells : A comparison of PH-domain-mediated methods with immunological methods. In: Biochemical Journal. 2008 ; Vol. 411, No. 2. pp. 441-448.
@article{9400d2438b094fa8a55f58c7b4041018,
title = "Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: A comparison of PH-domain-mediated methods with immunological methods",
abstract = "Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P 3 production using a specific monoclonal anti-PtdIns(3,4,5)P 3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3 production, measured by the membrane translocation of an epitope-tagged BTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3 hydrolysis by measuring the decay of the PtdIns(3,4,5)P3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P 3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3 in vitro. These data suggest that anti-PtdIns(3,4,5)P3 antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.",
keywords = "Carcinoma cell, Epidermal growth factor (EGF), Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P], Phosphoinositide, Phosphoinositide 3-kinase (PI3K), Pleckstrin homology domain (PH domain)",
author = "Yip, {Shu Chin} and Eddy, {Robert J.} and Branch, {Angie M.} and Huan Pang and Haiyan Wu and Ying Yan and Drees, {Beth E.} and Neilsen, {Paul O.} and Condeelis, {John S.} and Backer, {Jonathan M.}",
year = "2008",
month = "4",
day = "15",
doi = "10.1042/BJ20071179",
language = "English (US)",
volume = "411",
pages = "441--448",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "2",

}

TY - JOUR

T1 - Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells

T2 - A comparison of PH-domain-mediated methods with immunological methods

AU - Yip, Shu Chin

AU - Eddy, Robert J.

AU - Branch, Angie M.

AU - Pang, Huan

AU - Wu, Haiyan

AU - Yan, Ying

AU - Drees, Beth E.

AU - Neilsen, Paul O.

AU - Condeelis, John S.

AU - Backer, Jonathan M.

PY - 2008/4/15

Y1 - 2008/4/15

N2 - Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P 3 production using a specific monoclonal anti-PtdIns(3,4,5)P 3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3 production, measured by the membrane translocation of an epitope-tagged BTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3 hydrolysis by measuring the decay of the PtdIns(3,4,5)P3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P 3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3 in vitro. These data suggest that anti-PtdIns(3,4,5)P3 antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

AB - Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P 3 production using a specific monoclonal anti-PtdIns(3,4,5)P 3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3 production, measured by the membrane translocation of an epitope-tagged BTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3 hydrolysis by measuring the decay of the PtdIns(3,4,5)P3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P 3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3 in vitro. These data suggest that anti-PtdIns(3,4,5)P3 antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

KW - Carcinoma cell

KW - Epidermal growth factor (EGF)

KW - Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P]

KW - Phosphoinositide

KW - Phosphoinositide 3-kinase (PI3K)

KW - Pleckstrin homology domain (PH domain)

UR - http://www.scopus.com/inward/record.url?scp=42449135838&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=42449135838&partnerID=8YFLogxK

U2 - 10.1042/BJ20071179

DO - 10.1042/BJ20071179

M3 - Article

C2 - 18215145

AN - SCOPUS:42449135838

VL - 411

SP - 441

EP - 448

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -