# Structural Biochemistry/Phosphatidylserine Binding Motif and PKC

PKC is controlled by diacylglycerol and the amino phospholipid, phosphatidylserine. The molecular basis for the phosphatidylserine specificity was proposed to arise from the presence of a putative phosphatidylserine binding motif, localized in the C2 domain of PKC. In order to determine whether this motif mediates the interaction of PKC with phosphatidylserine, the carboxyl-terminal basic residues were mutated to Alanine in PKC BII. Additionally, the phosphatidylserine regulation of the mutant enzyme was observed.
Membrane binding and activity measurements revealed that the phosphatidylserine regulation for the mutant protein was indistinguishable from that of wild-type protein kinase C. Neither the apparent membrane affinity for phosphatidylserine-containing membranes in the presence or absence of diacylglycerol nor the phsphatidyl-serine-dependence for activation was affected by removal of the conserved basic residues at the carboxyl terminus of the consensus sequence.
The protein kinase C family of serine/threonine kinases transduces the multitude of extracellular signals that result in generation of the lipid second messenger, diacylglycerol. This lipid allows protein kinase C to translocate from the cytosol to the plasma membrane where it becomes activated by an additional interaction with phosphatidylserine. This is mediated by two membrane-targeting modules: the C1 and C2 domains. One of the methods to observe this process is through mutagenesis. With this, mutation of Lys 236 and Arg 238 to Alanine occurs in PKC BII. This is achieved utilizing PCR with wild-type PKC BII in pBlueScript as a template.
Another method used was Protein Expression and Cell Fractionation. In this process, recombinant baculovirus encoding wild-type or K236A/R238A PKC BII was incubated with Sf-21 cells for 4 hours and then diluted with media and incubated at 27 degrees Celsius.
Another method used was Western Blot Analysis of Expressed Protein Kinase C. In this method, the distribution of PKC in the detergent-soluble and detergent-insoluble fractions was analyzed by Western blot analysis. Samples of the extracts were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane.
Another method used was through lipid observation in which sucrose-loaded large unilamellar vesicles were used and prepared by drying mixtures of lipids in chloroform under a stream of nitrogen, followed by evacuation under vacuum, suspension of lipids in 20 mM HEPES, pH 7.5, 170 mM sucrose, and then 5 freeze-thaw cycles followed by extrusion using a Liposofast microextruder.
Another method used was the Protein Kinase C Activity Assay and the Protein Kinase C Membrane-binding Assay in which its membrane affinity was determined by measuring the binding to sucrose-loaded vesicles. The fraction of sedimented was determined by assaying the activity of PKC in the supernatant and pellet under identical conditions using the cofactor-independent substrate, protamine sulfate. In this case, the membrane affinity was calculated as the ratio of free/bound protein kinase C divided by the total lipid concentration. Lastly, Data Analysis was another technique used for observation. In this technique, free calcium was calculated using a program that takes into account concentrations of Mg2+, ATP, Na+, EGTA, EDTA, and pH.
Reference: Joanne E. Johnson, Amelia S. Edwards, and Alexandra Newton from Departments of Pharmacology and Chemistry and Biochemistry, University of California, San Diego