Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles.

The specificity of the phospholipid cofactor requirement of rat brain protein kinase C was investigated using Triton X-100 mixed micellar methods. Sixteen analogues of phosphatidylserine were prepared and tested for their ability to support protein kinase C activity, [3H]phorbol 12,13-dibutyrate binding, and protein kinase C binding to mixed micelles. Phosphatidylserinol, -L-serine methyl ester, -N-acetyl-L-serine, -2-hydroxyacetate, -3-hydroxypropionate, and -4-hydroxybutyrate did not activate protein kinase C in mixed micelles containing 2 mol % of sn-1,2-dioleoylglycerol. This indicates that both the carboxyl and amino moieties are important for activation. Phosphatidyl-D-serine and -L-homoserine were incapable of supporting full activation; this demonstrates stereospecificity and the importance of the distance between the phosphate and carboxyl and amino moieties. Since 1,2-rac-phosphatidyl-L-serine and 1,3-phosphatidyl-L-serine fully supported protein kinase C activity, the stereochemistry within the glycerol backbone at the interface was not necessary for maximal activation. Neither lysophosphatidyl-L-serine nor 1-oleoyl-2-acetyl-sn-glycero-3-phospho-L-serine supported protein kinase C activity implying that the interfacial conformation is critical to the activation process. The phospholipid dependencies of [3H]phorbol 12,13-dibutyrate binding and of protein kinase C binding to mixed micelles containing sn-1,2-dioleoylglycerol did not mirror those for activation. The data demonstrate that protein kinase C possesses a high degree of specificity with respect to phospholipid activation and implicate several functional groups within the phospho-L-serine polar head group in binding and activation.