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Point Mutations in the Split PLC-γ1 PH Domain Modulate Phosphoinositide Binding

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Author(s)
Sung-Kuk KimSung-Mo WeeJong-Soo ChangTaeg-Kyu KwonDo Sik MinYoung Han LeePann-Ghill Suh
Keimyung Author(s)
Kwon, Taeg Kyu
Department
Dept. of Immunology (면역학)
Journal Title
Journal of Biochemistry & Molecular Biology
Issued Date
2004
Volume
37
Issue
6
Keyword
Dot-blottingPhosphatidylinositol 4,5-bisphosphatePhospholipase C-γ1Pleckstrin homology domainProteinphosphoinositide interaction
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 -terminal (PH1) 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)P2, 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 PH2 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.
Keimyung Author(s)(Kor)
권택규
Publisher
School of Medicine
Citation
Sung-Kuk Kim et al. (2004). Point Mutations in the Split PLC-γ1 PH Domain Modulate Phosphoinositide Binding. Journal of Biochemistry & Molecular Biology, 37(6), 720–725. doi: 10.5483/BMBRep.2004.37.6.720
Type
Article
ISSN
1225-8687
DOI
10.5483/BMBRep.2004.37.6.720
URI
https://kumel.medlib.dsmc.or.kr/handle/2015.oak/33730
Appears in Collections:
1. School of Medicine (의과대학) > Dept. of Immunology (면역학)
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