Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.
We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 binding sites in four phospholipase C (PLC) isozymes (delta1, beta1, beta2, and beta3), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P3, D-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta1 was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated delta1-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P3 to PLC-delta1 indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P3 to PLC-beta1, -beta2, or -beta3 was not detected. The catalytic activity of PLC-delta1 was inhibited by the product D-Ins(1,4,5)P3, whereas no inhibition of PLC-beta1, -beta2, or -beta3 activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2 binding site of PLC-delta1 and that a similar site is not present in PLC-beta1, -beta2, or -beta3. The data are consistent with the idea that the PH domain of PLC-delta1, but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2 and is subject to product inhibition.
Tall
E
E
Department of Anesthesiology, State University of New York at Stony Brook, 11794, USA.
Dormán
G
G
Garcia
P
P
Runnels
L
L
Shah
S
S
Chen
J
J
Profit
A
A
Gu
Q M
QM
Chaudhary
A
A
Prestwich
G D
GD
Rebecchi
M J
MJ
D001577:Benzophenones / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D054799:Phospholipase C beta; D054803:Phospholipase C delta; D010777:Photochemistry; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*
C106618:1-O-(3-(4-benzoyldihydrocinnamidiyl)propyl)-myoinositol 1,4,5-trisphosphate; D001577:Benzophenones; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; C518036:PLCB2 protein, human; C518039:PLCB3 protein, human; C518024:PLCD1 protein, human; D054799:Phospholipase C beta; D054803:Phospholipase C delta