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Point mutation in the split PLC-gamma1 PH domain modulate phosphoinositide binding.

Title
Point mutation in the split PLC-gamma1 PH domain modulate phosphoinositide binding.
Author
이영한
Issue Date
2004-11
Publisher
SPRINGER-VERLAG SINGAPORE PTE LTD
Citation
JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, v.37, Issue.6, Page.720-725
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.
URI
https://www.scopus.com/record/display.uri?eid=2-s2.0-13544264660&origin=inward&txGid=71978be17783b99cbf84360e845456cahttps://repository.hanyang.ac.kr/handle/20.500.11754/154820
ISSN
1225-8687
Appears in Collections:
COLLEGE OF SCIENCE AND CONVERGENCE TECHNOLOGY[E](과학기술융합대학) > ETC
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