Phosphatidylinositol (3,4,5)- trisphosphate specifically intracts with the phox homology domain of phospholipase D1 and stimulates its activity

Jun Sung Lee, Jong Hyun Kim, Il Ho Jang, Hyeon Soo Kim, Jung Min Han, Andrius Kazlauskas, Hitoshi Yagisawa, Pann Ghill Suh, Sung Ho Ryu

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays key roles in cellular signal transduction by mediating extracellular stimuli including hormones, growth factors, neurotransmitters, cytokines and extracellular matrix molecules. The molecular mechanisms by which domains regulate the activity of PLD - especially the phox homology (PX) domain - have not been fully elucidated. In this study, we have examined the properties of the PX domains of PLD1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Interestingly, the PX domain of PLD1, but not that of PLD2, was found to specifically interact with phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). We found that mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine (R179A or R179K, respectively) disrupts PtdIns(3,4,5)P3 binding. In NIH-3T3 cells, the EGFP-PLD1 PX wild-type domain, but not the two mutants, localized to the plasma membrane after 5-minute treatment with platelet-derived growth factor (PDGF). The enzymatic activity of PLD1 was stimulated by adding PtdIns(3,4,5)P3 in vitro. Treatment with PDGF resulted in the significant increase of PLD1 activity and phosphorylation of the downstream extracellular signal-regulated kinases (ERKs), which was blocked by pre-treatment of HEK 293 cells with phosphoinositide 3-kinase (PI3K) inhibitor after the endogenous PLD2 had been depleted by siRNA specific for PLD2. Nevertheless, both PLD1 mutants (which cannot interact with PtdIns(3,4,5)P3) did not respond to treatment with PDGF. Moreover, PLD1 was activated in HepG2 cells stably expressing the Y40/51 mutant of PDGF receptor that is required for the binding with PI3K. Our results suggest that the PLD1 PX domain enables PLD1 to mediate signal transduction via ERK1/2 by providing a direct binding site for PtdIns(3,4,5)P3 and by activating PLD1.

Original languageEnglish
Pages (from-to)4405-4413
Number of pages9
JournalJournal of Cell Science
Volume118
Issue number19
DOIs
Publication statusPublished - 2005 Oct 1
Externally publishedYes

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Phospholipase D
Platelet-Derived Growth Factor
1-Phosphatidylinositol 4-Kinase
Signal Transduction
Platelet-Derived Growth Factor Receptors
NIH 3T3 Cells
Phosphatidic Acids
HEK293 Cells
Extracellular Signal-Regulated MAP Kinases
Hep G2 Cells
Phosphatidylinositols
Choline
Phosphatidylcholines
Alanine
Small Interfering RNA
Lysine
Extracellular Matrix
Neurotransmitter Agents
Arginine
phosphatidylinositol 3,4,5-triphosphate

Keywords

  • ERK phosphorylation
  • PDGF
  • Phosphoinositides
  • PLD
  • PX domain

ASJC Scopus subject areas

  • Cell Biology

Cite this

Phosphatidylinositol (3,4,5)- trisphosphate specifically intracts with the phox homology domain of phospholipase D1 and stimulates its activity. / Lee, Jun Sung; Kim, Jong Hyun; Jang, Il Ho; Kim, Hyeon Soo; Han, Jung Min; Kazlauskas, Andrius; Yagisawa, Hitoshi; Suh, Pann Ghill; Ryu, Sung Ho.

In: Journal of Cell Science, Vol. 118, No. 19, 01.10.2005, p. 4405-4413.

Research output: Contribution to journalArticle

Lee, Jun Sung ; Kim, Jong Hyun ; Jang, Il Ho ; Kim, Hyeon Soo ; Han, Jung Min ; Kazlauskas, Andrius ; Yagisawa, Hitoshi ; Suh, Pann Ghill ; Ryu, Sung Ho. / Phosphatidylinositol (3,4,5)- trisphosphate specifically intracts with the phox homology domain of phospholipase D1 and stimulates its activity. In: Journal of Cell Science. 2005 ; Vol. 118, No. 19. pp. 4405-4413.
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abstract = "Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays key roles in cellular signal transduction by mediating extracellular stimuli including hormones, growth factors, neurotransmitters, cytokines and extracellular matrix molecules. The molecular mechanisms by which domains regulate the activity of PLD - especially the phox homology (PX) domain - have not been fully elucidated. In this study, we have examined the properties of the PX domains of PLD1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Interestingly, the PX domain of PLD1, but not that of PLD2, was found to specifically interact with phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). We found that mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine (R179A or R179K, respectively) disrupts PtdIns(3,4,5)P3 binding. In NIH-3T3 cells, the EGFP-PLD1 PX wild-type domain, but not the two mutants, localized to the plasma membrane after 5-minute treatment with platelet-derived growth factor (PDGF). The enzymatic activity of PLD1 was stimulated by adding PtdIns(3,4,5)P3 in vitro. Treatment with PDGF resulted in the significant increase of PLD1 activity and phosphorylation of the downstream extracellular signal-regulated kinases (ERKs), which was blocked by pre-treatment of HEK 293 cells with phosphoinositide 3-kinase (PI3K) inhibitor after the endogenous PLD2 had been depleted by siRNA specific for PLD2. Nevertheless, both PLD1 mutants (which cannot interact with PtdIns(3,4,5)P3) did not respond to treatment with PDGF. Moreover, PLD1 was activated in HepG2 cells stably expressing the Y40/51 mutant of PDGF receptor that is required for the binding with PI3K. Our results suggest that the PLD1 PX domain enables PLD1 to mediate signal transduction via ERK1/2 by providing a direct binding site for PtdIns(3,4,5)P3 and by activating PLD1.",
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AU - Han, Jung Min

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AB - Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays key roles in cellular signal transduction by mediating extracellular stimuli including hormones, growth factors, neurotransmitters, cytokines and extracellular matrix molecules. The molecular mechanisms by which domains regulate the activity of PLD - especially the phox homology (PX) domain - have not been fully elucidated. In this study, we have examined the properties of the PX domains of PLD1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Interestingly, the PX domain of PLD1, but not that of PLD2, was found to specifically interact with phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). We found that mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine (R179A or R179K, respectively) disrupts PtdIns(3,4,5)P3 binding. In NIH-3T3 cells, the EGFP-PLD1 PX wild-type domain, but not the two mutants, localized to the plasma membrane after 5-minute treatment with platelet-derived growth factor (PDGF). The enzymatic activity of PLD1 was stimulated by adding PtdIns(3,4,5)P3 in vitro. Treatment with PDGF resulted in the significant increase of PLD1 activity and phosphorylation of the downstream extracellular signal-regulated kinases (ERKs), which was blocked by pre-treatment of HEK 293 cells with phosphoinositide 3-kinase (PI3K) inhibitor after the endogenous PLD2 had been depleted by siRNA specific for PLD2. Nevertheless, both PLD1 mutants (which cannot interact with PtdIns(3,4,5)P3) did not respond to treatment with PDGF. Moreover, PLD1 was activated in HepG2 cells stably expressing the Y40/51 mutant of PDGF receptor that is required for the binding with PI3K. Our results suggest that the PLD1 PX domain enables PLD1 to mediate signal transduction via ERK1/2 by providing a direct binding site for PtdIns(3,4,5)P3 and by activating PLD1.

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