Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy

Stephen A Watt, Wendy A Kimber, Ian Neil Fleming, Nick R Leslie, C Peter Downes, John M Lucocq

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.
Original languageEnglish
Pages (from-to)653-663
Number of pages11
JournalBiochemical Journal
Volume377
Issue number3
DOIs
Publication statusPublished - 1 Feb 2004

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Immunoelectron Microscopy
Phosphatidylinositols
Microscopic examination
Proteins
Lipids
Labeling
PTEN Phosphohydrolase
Null Lymphocytes
Chromosomes, Human, Pair 10
phosphatidylinositol 3,4-diphosphate
Pleckstrin Homology Domains
platelet protein P47
Endosomes
Platelet-Derived Growth Factor
Phosphatases
Second Messenger Systems
Cell membranes
Chromosomes
Glutathione Transferase
Phosphoric Monoester Hydrolases

Keywords

  • animals
  • blood proteins
  • carrier proteins
  • cell line
  • tumor cell line
  • DNA probes
  • down-regulation
  • endoplasmic reticulum
  • humans
  • hydrogen peroxide
  • intracellular membranes
  • intracellular signaling peptides and proteins
  • intracellular space
  • membrane proteins
  • mice
  • immunoelectron microscopy
  • PTEN phosphohydrolase
  • peptides
  • phosphatidylinositol phosphates
  • phosphoproteins
  • phosphoric monoester hydrolases
  • platelet-derived growth factor
  • tertiary protein structure
  • sequence homology, amino acid
  • staining and labeling
  • swiss 3T3 cells
  • tumor suppressor proteins

Cite this

Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy. / Watt, Stephen A; Kimber, Wendy A; Fleming, Ian Neil; Leslie, Nick R; Downes, C Peter; Lucocq, John M.

In: Biochemical Journal, Vol. 377, No. 3, 01.02.2004, p. 653-663.

Research output: Contribution to journalArticle

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abstract = "PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.",
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T1 - Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy

AU - Watt, Stephen A

AU - Kimber, Wendy A

AU - Fleming, Ian Neil

AU - Leslie, Nick R

AU - Downes, C Peter

AU - Lucocq, John M

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AB - PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.

KW - animals

KW - blood proteins

KW - carrier proteins

KW - cell line

KW - tumor cell line

KW - DNA probes

KW - down-regulation

KW - endoplasmic reticulum

KW - humans

KW - hydrogen peroxide

KW - intracellular membranes

KW - intracellular signaling peptides and proteins

KW - intracellular space

KW - membrane proteins

KW - mice

KW - immunoelectron microscopy

KW - PTEN phosphohydrolase

KW - peptides

KW - phosphatidylinositol phosphates

KW - phosphoproteins

KW - phosphoric monoester hydrolases

KW - platelet-derived growth factor

KW - tertiary protein structure

KW - sequence homology, amino acid

KW - staining and labeling

KW - swiss 3T3 cells

KW - tumor suppressor proteins

U2 - 10.1042/BJ20031397

DO - 10.1042/BJ20031397

M3 - Article

VL - 377

SP - 653

EP - 663

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -