Protein kinase C isoenzymes display differential affinity for phorbol esters. Analysis of phorbol ester receptors in B cell differentiation.
Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.
Marquez
C
C
Centro de Biología Molecular, CSIC, Madrid, Spain.
Martinez
C
C
Kroemer
G
G
Bosca
L
L
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000502:physiology; D002352:Carrier Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000378:metabolism* / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011955:Receptors, Drug / Q000032:analysis*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D007527:Isoenzymes; D010703:Phorbol Esters; D012333:RNA, Messenger; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C; D002118:Calcium