Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.
Biochemistry
; 36(23): 7239-48, 1997 Jun 10.
Article
en En
| MEDLINE
| ID: mdl-9188725
ABSTRACT
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fosfolipasas de Tipo C
/
Fosfatos de Fosfatidilinositol
/
Fosfatidilinositol 4,5-Difosfato
/
Reactivos de Enlaces Cruzados
/
Isoenzimas
Límite:
Humans
Idioma:
En
Revista:
Biochemistry
Año:
1997
Tipo del documento:
Article
País de afiliación:
Estados Unidos