Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-δ1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane

Hitoshi Yagisawa, Kaori Sakuma, Hugh F. Paterson, Robert Cheung, Victoria Allen, Hajime Hirata, Yutaka Watanabe, Masato Hirata, Roger L. Williams, Matilda Katan

Research output: Contribution to journalArticle

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Abstract

The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-δ1 (PLC-δ1) binds to both D-myo-inositol 1,4,5- trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun, 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-δ1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, add Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-δ1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, add measurements of phospholipase activity using PtdIns- (4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-δ1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P3 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-δ1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.

Original languageEnglish
Pages (from-to)417-424
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number1
DOIs
Publication statusPublished - Jan 2 1998

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Basic Amino Acids
Phospholipases
Type C Phospholipases
Cell membranes
Phosphatidylinositol 4,5-Diphosphate
Cell Membrane
Ligands
Hydrolysis
Mutation
Phosphatidylinositols
Membranes
Phospholipids
Acidic Amino Acids
Phosphoinositide Phospholipase C
Phosphatidylinositol Phosphates
Mutagenesis
Inositol 1,4,5-Trisphosphate
Microinjections
Enzyme activity
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-δ1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane. / Yagisawa, Hitoshi; Sakuma, Kaori; Paterson, Hugh F.; Cheung, Robert; Allen, Victoria; Hirata, Hajime; Watanabe, Yutaka; Hirata, Masato; Williams, Roger L.; Katan, Matilda.

In: Journal of Biological Chemistry, Vol. 273, No. 1, 02.01.1998, p. 417-424.

Research output: Contribution to journalArticle

Yagisawa, Hitoshi ; Sakuma, Kaori ; Paterson, Hugh F. ; Cheung, Robert ; Allen, Victoria ; Hirata, Hajime ; Watanabe, Yutaka ; Hirata, Masato ; Williams, Roger L. ; Katan, Matilda. / Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-δ1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 1. pp. 417-424.
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abstract = "The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-δ1 (PLC-δ1) binds to both D-myo-inositol 1,4,5- trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun, 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-δ1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, add Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-δ1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, add measurements of phospholipase activity using PtdIns- (4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-δ1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P3 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-δ1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.",
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AU - Yagisawa, Hitoshi

AU - Sakuma, Kaori

AU - Paterson, Hugh F.

AU - Cheung, Robert

AU - Allen, Victoria

AU - Hirata, Hajime

AU - Watanabe, Yutaka

AU - Hirata, Masato

AU - Williams, Roger L.

AU - Katan, Matilda

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N2 - The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-δ1 (PLC-δ1) binds to both D-myo-inositol 1,4,5- trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun, 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-δ1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, add Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-δ1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, add measurements of phospholipase activity using PtdIns- (4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-δ1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P3 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-δ1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.

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