Characterization of putative polyphosphoinositide binding motifs from phospholipase C beta 2.
Several phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-regulated actin-binding proteins and most phosphoinositide-specific phospholipases C (PI-PLCs) comprise a basic amino acid motif (KxxxKxKK, where x denotes any amino acid), which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have shown 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 [Simões et al. (1993) FEBS Lett. 331, 248]. Here, we present a detailed analysis of the effects of various peptides related to peptide P1 aimed at understanding the mechanisms of peptide-mediated PLC beta 2 stimulation. Peptide KILIKNKK (P2), which comprises only the basic amino acid consensus motif, also stimulated PLC beta 2, although higher concentrations were 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 beta 2. Peptide KILIKNKKQFSGPTSS (P4), which includes the nine amino acids flanking the carboxy-terminus of the KILIKNKK motif within the sequence 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 PtdInsP2 but not in the presence of other phospholipids including phosphatidylinositol 4-phosphate. The results suggest that the basic amino acid sequence physically interacts with PtdInsP2.(ABSTRACT TRUNCATED AT 250 WORDS)
Simões
A P
AP
German Cancer Research Center, Heidelberg, Germany.
Reed
J
J
Schnabel
P
P
Camps
M
M
Gierschik
P
P
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D054799:Phospholipase C beta; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C518036:PLCB2 protein, human; D054799:Phospholipase C beta