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Suh, Pann-Ghill
BioSignal Network Lab (BSN)
Research Interests
  • Signal transduction, cancer, metabolism, phospholipase C

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Point mutations in the split PLC-gamma 1 PH domain modulate phosphoinositide binding

Cited 4 times inthomson ciCited 4 times inthomson ci
Title
Point mutations in the split PLC-gamma 1 PH domain modulate phosphoinositide binding
Author
Kim, SKWee, SMChang, JSKwon, TKMin, DSLee, YHSuh, Pann-Ghill
Keywords
Dot-blotting; Phosphatidylinositol 4,5-bisphosphate; Phospholipase C-γ1; Pleckstrin homology domain; Protein-phosphoinositide interaction
Issue Date
200411
Publisher
Springer Verlag
Citation
JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, v.37, no.6, pp.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.
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ISSN
1225-8687
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