Interaction of elongation factor-1α and pleckstrin homology domain of phospholipase C-γ1 with activating its activity

Jong Soo Chang, Heon Seok, Taeg Kyu Kwon, Do Sik Min, Bong Hyun Ahn, Young Han Lee, Ju Won Suh, Jong Woo Kim, Shintaro Iwashita, Akira Omori, Sachiyo Ichinose, Osamu Numata, Jeong Kon Seo, Yong Seok Oh, Pann Ghill Suh

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH2-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-γ1 with translational elongation factor (EF)-1α, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-γ1 followed by peptide sequence analysis, we identified EF-1α as a binding partner of a split PH domain of PLC-γ1. Analysis by site-directed mutagenesis of the PH domain revealed that the β2-sheet of a split PH domain is critical for the interaction with EF-1α. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP2). So the PH domain of PLC-γ1 binds to both EF-1α and PlP2. The binding affinity of EF-1α to the GST·PH domain fusion protein increased in the presence of PIP2, although PIP2 does not bind to EF-1α directly. This suggests that EF-1α may control the binding affinity between the PH domain and PIP2. PLC-γ1 is substantially activated in the presence of EF-1α with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-γ1 (Y509A/F510A) that lost its binding affinity to EF-1α shows basal level activity. Taken together, our data show that EF-1α plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-γ1 activity via a split PH domain.

Original languageEnglish
Pages (from-to)19697-19702
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number22
DOIs
Publication statusPublished - 2002 May 31

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Peptide Elongation Factor 1
Type C Phospholipases
Phosphatidylinositols
platelet protein P47
Pleckstrin Homology Domains
Fusion reactions
Cell signaling
Mutagenesis
1-Phosphatidylinositol 4-Kinase
Protein Sequence Analysis
Site-Directed Mutagenesis
Cell Extracts
Glutathione Transferase
Metabolism
Assays
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Chang, Jong Soo ; Seok, Heon ; Kwon, Taeg Kyu ; Min, Do Sik ; Ahn, Bong Hyun ; Lee, Young Han ; Suh, Ju Won ; Kim, Jong Woo ; Iwashita, Shintaro ; Omori, Akira ; Ichinose, Sachiyo ; Numata, Osamu ; Seo, Jeong Kon ; Oh, Yong Seok ; Suh, Pann Ghill. / Interaction of elongation factor-1α and pleckstrin homology domain of phospholipase C-γ1 with activating its activity. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 22. pp. 19697-19702.
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abstract = "The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH2-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-γ1 with translational elongation factor (EF)-1α, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-γ1 followed by peptide sequence analysis, we identified EF-1α as a binding partner of a split PH domain of PLC-γ1. Analysis by site-directed mutagenesis of the PH domain revealed that the β2-sheet of a split PH domain is critical for the interaction with EF-1α. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP2). So the PH domain of PLC-γ1 binds to both EF-1α and PlP2. The binding affinity of EF-1α to the GST·PH domain fusion protein increased in the presence of PIP2, although PIP2 does not bind to EF-1α directly. This suggests that EF-1α may control the binding affinity between the PH domain and PIP2. PLC-γ1 is substantially activated in the presence of EF-1α with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-γ1 (Y509A/F510A) that lost its binding affinity to EF-1α shows basal level activity. Taken together, our data show that EF-1α plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-γ1 activity via a split PH domain.",
author = "Chang, {Jong Soo} and Heon Seok and Kwon, {Taeg Kyu} and Min, {Do Sik} and Ahn, {Bong Hyun} and Lee, {Young Han} and Suh, {Ju Won} and Kim, {Jong Woo} and Shintaro Iwashita and Akira Omori and Sachiyo Ichinose and Osamu Numata and Seo, {Jeong Kon} and Oh, {Yong Seok} and Suh, {Pann Ghill}",
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Chang, JS, Seok, H, Kwon, TK, Min, DS, Ahn, BH, Lee, YH, Suh, JW, Kim, JW, Iwashita, S, Omori, A, Ichinose, S, Numata, O, Seo, JK, Oh, YS & Suh, PG 2002, 'Interaction of elongation factor-1α and pleckstrin homology domain of phospholipase C-γ1 with activating its activity', Journal of Biological Chemistry, vol. 277, no. 22, pp. 19697-19702. https://doi.org/10.1074/jbc.M111206200

Interaction of elongation factor-1α and pleckstrin homology domain of phospholipase C-γ1 with activating its activity. / Chang, Jong Soo; Seok, Heon; Kwon, Taeg Kyu; Min, Do Sik; Ahn, Bong Hyun; Lee, Young Han; Suh, Ju Won; Kim, Jong Woo; Iwashita, Shintaro; Omori, Akira; Ichinose, Sachiyo; Numata, Osamu; Seo, Jeong Kon; Oh, Yong Seok; Suh, Pann Ghill.

In: Journal of Biological Chemistry, Vol. 277, No. 22, 31.05.2002, p. 19697-19702.

Research output: Contribution to journalArticle

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T1 - Interaction of elongation factor-1α and pleckstrin homology domain of phospholipase C-γ1 with activating its activity

AU - Chang, Jong Soo

AU - Seok, Heon

AU - Kwon, Taeg Kyu

AU - Min, Do Sik

AU - Ahn, Bong Hyun

AU - Lee, Young Han

AU - Suh, Ju Won

AU - Kim, Jong Woo

AU - Iwashita, Shintaro

AU - Omori, Akira

AU - Ichinose, Sachiyo

AU - Numata, Osamu

AU - Seo, Jeong Kon

AU - Oh, Yong Seok

AU - Suh, Pann Ghill

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N2 - The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH2-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-γ1 with translational elongation factor (EF)-1α, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-γ1 followed by peptide sequence analysis, we identified EF-1α as a binding partner of a split PH domain of PLC-γ1. Analysis by site-directed mutagenesis of the PH domain revealed that the β2-sheet of a split PH domain is critical for the interaction with EF-1α. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP2). So the PH domain of PLC-γ1 binds to both EF-1α and PlP2. The binding affinity of EF-1α to the GST·PH domain fusion protein increased in the presence of PIP2, although PIP2 does not bind to EF-1α directly. This suggests that EF-1α may control the binding affinity between the PH domain and PIP2. PLC-γ1 is substantially activated in the presence of EF-1α with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-γ1 (Y509A/F510A) that lost its binding affinity to EF-1α shows basal level activity. Taken together, our data show that EF-1α plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-γ1 activity via a split PH domain.

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