title: Mutational analysis of a putative polyphosphoinositide binding site in phospholipase C-beta 2.
abstract: The phosphatidylinositol 4,5-bisphosphate (PtdIns-P2)-regulated actin-binding protein gelsolin and most phosphoinositide-specific phospholipases C (PLCs) comprise a basic amino acid motif ((K/R)xxxKxK(K/R); x denotes any amino acid) which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have challenged this hypothesis for PLC beta 2 by replacing one or several residues of this motif (KILIKNKK; residues 457-464) and examining the functional consequences of these alterations. The results show that the integrity of the basic motif is important for PtdInsP2 hydrolysis by PLC beta 2. Replacement of lysines 463 or 461 by arginine led to reduction or complete loss, respectively, of enzyme activity. The results provide further support to the concept that the function of the basic motif within the various PLCs is to bind the enzyme substrate PtdInsP2.
authors.affiliation: German Cancer Research Center, Heidelberg.
authors.forename: A P
authors.initials: AP
authors.lastname: Simões
authors.forename: M
authors.initials: M
authors.lastname: Camps
authors.forename: P
authors.initials: P
authors.lastname: Schnabel
authors.forename: P
authors.initials: P
authors.lastname: Gierschik
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase