Pleckstrin homology domain of phospholipase C-δ1: Its importance in lns(1,4,5)P3 / Ptdlns(4,5)P2 binding, enzymatic activity and translocation

H. Tegisawa, K. Sakuma, Y. Tamaoka, T. Mizobata, M. Fujii, H. Kamata, M. Hirata, H. Hirata

Research output: Contribution to journalArticlepeer-review

Abstract

Phospholipase C (PLC)-δl shows high D-myo-inositol 1 4,5-Irisphosphate (lns(1,4,5)P3) binding, which causes suppression of the enzyme. We have previously found the binding domain in the N-terminal PH (pleckstrin homology) domain of the enzyme and have shown phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) competes with lns(1,4,5)P3 on this site. We have also shown that an antibody raised against a synthetic peptide corresponding to residues 30-43 of PLC-δ1, which is rich in basic amino acids and binds lns(1,4,5)P3, blocks the binding and the catalytic activity. In this study, we examined the relevance of basic amino acids in the lns(1,4,5)P3/Ptdlns(4,5)P2 binding and the enzymatic activity by sitedirected mutagenesis, in which each basic amino acid in the N-terminal 60 residues was replaced to a neutral or an acidic amino acid. Replacement of K30, K32, R37, R40 or K43 significantly attenuated [3H]-lns(1,4,5)P3 binding, affinity for immobilized lns(1,4,5)P3, binding of Ptdlns(4,5)P2-containing liposomes and the catalytic activity, suggesting each of the basic residues is important for membrane targeting and regulation of the catalysis. Although PLC-S1 is essentially in cytoplasm as judged by both biochemical and immuno-histochemical analyses of NRK cells, there is a small fraction associated with the plasma membrane. When serumdeprived NRK cells were cultured in serum-rich (10% PCS) medium for eight hours, the amount of membrane-associated PLC-δl was increased to 3- to 5-fold. The PH domain of PLC-δ1 is likely to play a role in this translocation.

Original languageEnglish
Pages (from-to)583S
JournalBiochemical Society Transactions
Volume24
Issue number4
Publication statusPublished - 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry

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