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 Condeelis, Jonathan M. Backer

Research output: Contribution to journalArticle

24 Scopus citations

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

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
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Quantification of PtdIns(3,4,5)P<sub>3</sub> dynamics in EGF-stimulated carcinoma cells: A comparison of PH-domain-mediated methods with immunological methods'. Together they form a unique fingerprint.

  • Cite this