CHARACTERIZATION OF PUTATIVE POLYPHOSPHOINOSITIDE BINDING MOTIFS FROMPHOSPHOLIPASE C-BETA(2)

Several phosphatidylinositol 4,5-bisphosphate (PtdInsP(2))-regulated a ctin-binding proteins and most phosphoinositide-specific phospholipase s C (PI-PLCs) comprise a basic amino acid motif (KxxxKxKK, where x den otes any amino acid), which was previously suggested to represent a Pt dInsP(2)-binding site commonly present in these proteins. We have show n earlier that a peptide corresponding to amino acids 448-464 of human PLC beta(2) (LPSPEDLRGKILIKNKK, peptide P1) markedly and specifically stimulated the activity of this enzyme [Sim (o) over tilde es et al. (1993) FEES Lett. 331, 248]. Here, we present a detailed analysis of t he effects of various peptides related to peptide P1 aimed at understa nding the mechanisms of peptide-mediated PLC beta(2) stimulation. Pept ide KILIKNKK (P2), which comprises only the basic amino acid consensus motif, also stimulated PLC beta(2), although higher concentrations we re required to observe this stimulatory effect. The effects of P1 and P2 were not additive, indicating that the two peptides affect PLC beta (2) activity via the same mechanism. Peptide LPSPEDLRG (P3), composed of the amino-terminal half of P1, did not affect the activity of PLC b eta(2). Peptide KILIKNKKQFSGPTSS (P4), which includes the nine amino a cids flanking the carboxy-terminus of the KILIKNKK motif within the se quence of PLC beta(2) stimulated the enzyme but was indistinguishable in potency from P2. Circular dichroism analysis revealed that peptide P1 changes its conformation in the presence of PtdInsP(2) but not in t he presence of other phospholipids including phosphatidylinositol 4-ph osphate. The results suggest that the basic amino acid sequence physic ally interacts with PtdInsP(2). The role of the sequence corresponding to P1 within the native PLC beta(2) enzyme may be to offer the substr ate to the actual catalytic residues in a configuration more favorable to hydrolysis.