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Title: Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}), can also activate PKC in the presence of phosphatidylserine (PS) and Ca{sup 2+} with a K{sub PIP{sub 2}} of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP{sub 2} and DG on PKC. Here, the authors investigate the effect of PIP{sub 2} on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP{sub 2} inhibited specific binding of ({sup 3}H)phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP{sub 2} than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP{sub 2} is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (K{sub d{prime}}) against PIP{sub 2} concentration was linear over a range of 0.01-1 mol % with a K{sub i} of 0.043more » mol % and confirmed the competitive nature of inhibition between PDBu and PIP{sub 2}. Competition between PIP{sub 2} and phorbol ester could be determined in a liposomal assay system also. These results indicate that PIP{sub 2}, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP{sub 2} is a primary activator of the enzyme.« less
Authors:
; ; ;  [1]
  1. (New York State Office of Mental Retardation and Developmental Disabilities, Staten Island (USA))
Publication Date:
OSTI Identifier:
5443476
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (USA); Journal Volume: 28:12
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ENZYME INDUCTION; INHIBITION; PHORBOL ESTERS; BIOLOGICAL EFFECTS; PHOSPHOTRANSFERASES; BIOCHEMICAL REACTION KINETICS; INOSITOLS; PHOSPHONIC ACID ESTERS; TRITIUM COMPOUNDS; CARBOHYDRATES; CARCINOGENS; ENZYMES; ESTERS; GENE REGULATION; HYDROGEN COMPOUNDS; KINETICS; MONOSACCHARIDES; ORGANIC COMPOUNDS; ORGANIC PHOSPHORUS COMPOUNDS; PHOSPHORUS-GROUP TRANSFERASES; REACTION KINETICS; SACCHARIDES; TRANSFERASES 550201* -- Biochemistry-- Tracer Techniques