Point mutations in the split PLC-γ1 PH domain modulate phosphoinositide binding

Sung Kuk Kim, Sung Mo Wee, Jong Soo Chang, Taeg Kyu Kwon, Do Sik Min, Young Han Lee, Pann Ghill Suh

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH2-terminal (PH 1) and a split PH domain (nPH2 and cPH2). We previously reported that the split PH domain of PLC-γ1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P 2, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-γ1 nPH2 domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-γ1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-γ1 molecules showed reduced PI(4,5)P2 hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH1 and nPH2 domains are responsible for membrane-targeted translocation of PLC-γ1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro500 and His503 are critical for binding of PLC-γ1 to one of its substrates, PI(4,5)P2 in the membrane.

Original languageEnglish
Pages (from-to)720-725
Number of pages6
JournalJournal of Biochemistry and Molecular Biology
Volume37
Issue number6
Publication statusPublished - 2004 Nov 30

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Type C Phospholipases
Phosphatidylinositols
Point Mutation
Amino Acids
Molecules
Membranes
Neutral Amino Acids
Emitter coupled logic circuits
Mutagenesis
Site-Directed Mutagenesis
Green Fluorescent Proteins
Pleckstrin Homology Domains
platelet protein P47
Alanine
Hydrolysis
Assays
Proteins
Fusion reactions
Substrates
Serum

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Kim, S. K., Wee, S. M., Chang, J. S., Kwon, T. K., Min, D. S., Lee, Y. H., & Suh, P. G. (2004). Point mutations in the split PLC-γ1 PH domain modulate phosphoinositide binding. Journal of Biochemistry and Molecular Biology, 37(6), 720-725.
Kim, Sung Kuk ; Wee, Sung Mo ; Chang, Jong Soo ; Kwon, Taeg Kyu ; Min, Do Sik ; Lee, Young Han ; Suh, Pann Ghill. / Point mutations in the split PLC-γ1 PH domain modulate phosphoinositide binding. In: Journal of Biochemistry and Molecular Biology. 2004 ; Vol. 37, No. 6. pp. 720-725.
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abstract = "A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH2-terminal (PH 1) and a split PH domain (nPH2 and cPH2). We previously reported that the split PH domain of PLC-γ1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P 2, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-γ1 nPH2 domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-γ1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-γ1 molecules showed reduced PI(4,5)P2 hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH1 and nPH2 domains are responsible for membrane-targeted translocation of PLC-γ1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro500 and His503 are critical for binding of PLC-γ1 to one of its substrates, PI(4,5)P2 in the membrane.",
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Kim, SK, Wee, SM, Chang, JS, Kwon, TK, Min, DS, Lee, YH & Suh, PG 2004, 'Point mutations in the split PLC-γ1 PH domain modulate phosphoinositide binding', Journal of Biochemistry and Molecular Biology, vol. 37, no. 6, pp. 720-725.

Point mutations in the split PLC-γ1 PH domain modulate phosphoinositide binding. / Kim, Sung Kuk; Wee, Sung Mo; Chang, Jong Soo; Kwon, Taeg Kyu; Min, Do Sik; Lee, Young Han; Suh, Pann Ghill.

In: Journal of Biochemistry and Molecular Biology, Vol. 37, No. 6, 30.11.2004, p. 720-725.

Research output: Contribution to journalArticle

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AU - Kim, Sung Kuk

AU - Wee, Sung Mo

AU - Chang, Jong Soo

AU - Kwon, Taeg Kyu

AU - Min, Do Sik

AU - Lee, Young Han

AU - Suh, Pann Ghill

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