Autophosphorylation of protein kinase C may require a high order of protein-phospholipid aggregates.
The activation of protein kinase C (PKC) usually displays cofactor requirements that include phosphatidylserine (PS), diacylglycerol, and calcium. A complicating factor is that good exogenous substrates of PKC are polycationic proteins or peptides that form aggregates with PS in the assay. This study examined the autophosphorylation of PKC using assays with phospholipid provided in the form of vesicles or phospholipid-Triton mixed micelles. The results showed a close correlation between PKC autophosphorylation and the formation of aggregated assay components. Aggregation occurred primarily by the action of Mg2+ on phospholipids and appeared to underlie a number of major features of PKC autophosphorylation. For example, autophosphorylation required higher concentrations of PS than phosphorylation of exogenous substrates. This appeared to be the result of the different PS requirements of aggregation by divalent metal ions and cationic substrates. An unanticipated result was that aggregation of mixed micelles showed specificity for PS, high cooperativity with respect to several agents, and a requirement for calcium. These parameters were remarkably similar to those describing PKC autophosphorylation. Several major implications are evident in this study. Since the autophosphorylation assay is not a well defined system of monodisperse materials, autophosphorylation of PKC may proceed by intra- or interpeptide mechanism. The uniform correlation between aggregation and production of PKC activity suggested that kinetic parameters may represent interactions of assay components other than the enzyme. Aggregation, which appeared necessary for in vitro activation of PKC, may represent the expression of important but undefined in vivo requirements for this enzyme's function.
Bazzi
M D
MD
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Nelsestuen
G L
GL
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008823:Micelles; D010743:Phospholipids / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D008823:Micelles; D010743:Phospholipids; D011493:Protein Kinase C; D008274:Magnesium