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

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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.

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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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.