Opposing effects of Protein Kinase A and C causes differential phosphatidyserine exposure in a CD47 receptor mediated erythrocyte apoptotic pathway

Erythrocyte apoptosis, like nucleated cell death, is characterised by phosphatidylserine exposure at the outer membrane leaflet. Our group has previously reported that ligation of a monoclonal antibody, BRIC 126 can mediate red cell apoptosis and subsequent PS exposure, through a CD47 receptor mediated pathway. We have also demonstrated that several membrane proteins are involved in this pathway and one such interaction is a direct protein: protein interaction between CD47 and protein 4.1R. Further studies have shown that red cells deficient in protein 4.1R undergo increased PS exposure in response to BRIC-126 than do normal cells, suggesting that protein 4.1R is critical to this pathway. In order to further elucidate the CD47 apoptotic pathway we have inhibited Protein Kinase A and Protein Kinase C whilst ligating CD47 with BRIC-126, as these kinases are known to interact with protein 4.1R at specific serine residues and are often implicated in cell signalling cascades, especially apoptosis. We have used flow cytometry in conjunction with an annexin V-FITC binding assay to compare mean percentage annexin V positive cells whilst inhibiting protein kinase A and protein kinase C using the cell permeable specific inhibitors Bisindolylmaleimide I, Go 6976 and KT 5720. Our findings suggest opposing effects of Protein kinase A and Protein Kinase C compared with control erythrocytes. We observed that PS exposure was decreased in cells with the specific cell permeable PKC inhibitors Bisindolylmaleimide I and Go 6976 present but increased in cells with the specific PKA cell permeable inhibitor KT5720 present. The increase in PS exposure when PKA inhibitor KT5720 is present is similar to the effect seen in individuals deficient in protein 4.1R. The difference in PS exposure between PKC inhibition and PKA inhibition suggests opposing effects of PKA and PKC during CD47 ligation and a possible interaction with protein 4.1R, this may also suggest that specific phosphorylation events on protein 4.1R are critical to the CD47 apoptotic pathway. Ongoing studies are investigating isoform specific inhibition of PKC and PKA along with 2D phosphoimmunoblots to look at changes in PI before and after ligation of BRIC 126