Protein Kinases Associated with Activation of Sickle Red Blood Cell Adhesion.

We have previously found that activation of adenylate cyclase in sickle red cells (SS RBCs) results in increased RBC adhesion to cultured endothelial cells (EC) through activation of protein kinase A (PKA). Protein tyrosine phosphorylation by src family kinases, known to be present in normal RBCs, is also involved in enhancing SS RBC adhesion to EC, as inhibition of these kinases also inhibits activation of adhesion. However, in normal RBCs neither activation of adenylate cyclase nor cAMP analogs elicits a similar increase in cell adhesion. We therefore investigated whether normal and SS RBCs differed with respect to the protein kinases downstream of cAMP, such as PKA catalytic and regulatory subunits and protein tyrosine kinases p56lck, p56lyn and p72syk. We also investigated the extracellular signal regulated kinases p44 and p42 (ERK 1 and 2 respectively), which can be activated downstream of tyrosine kinases. PKA catalytic and regulatory subunits and ERK1/2 were studied after SS and normal RBCs were sham-treated or treated for 30 min with 80 μM forskolin, which activates adenylate cyclase. For protein tyrosine kinases, both SS and normal RBCs were sham-treated or treated with 1μg/ml Pertussis toxin, which suppresses Gαi-mediated inhibition of adenylate cyclase. Equal amounts of total membrane proteins were separated by polyacrylamide gel electrophoresis before transfer to nitrocellulose membrane. Protein kinases were detected in isolated membranes by immunoblotting using specific antibodies. Immunoblots showed that the levels of membrane-associated PKAcβ subunit were similar in both SS and normal RBCs and did not change after forskolin stimulation. However, treatment of SS RBCs with forskolin induced a significant reduction in membrane-bound PKARIIβ subunit. In general, dissociation of PKAR (regulatory) subunits is associated with activation of the catalytic (c) domain. In contrast, no change in the amount of membrane-bound PKARIIβ was observed when normal RBCs were treated with forskolin. These data demonstrate that PKARIIβ dissociated from PKAcβ after adenylate cyclase activation in SS RBCs, consistent with activation of PKAcβ. Our results thus suggest that activation of PKAcβ might be one of the mechanisms contributing to increased SS RBC adhesion. The amount of membrane-bound PKARIIα in SS RBCs did not change with stimulation, suggesting that PKAcα may not undergo activation as a result of forskolin stimulation. Immunoblots also showed no change in the levels of membrane-bound p56lck, p56lyn and p72syk detected before and after normal or SS RBCs were stimulated with Pertussis toxin. However, normal RBCs contained lower levels of protein tyrosine kinases, including p56lyn, p56lck and p76syk. We also found that ERK1/2 are bound to the membrane in normal and SS RBCs. The total amount of membrane-associated ERK1 (p44) is markedly higher in SS RBCs than in normal RBCs, and membrane ERK1 levels increased only in SS RBCs after stimulation with forskolin. Taken together, our results suggest that PKAcβ is activated in SS RBCs but not in normal RBCs in response to elevation of cAMP. The higher levels of src kinases in SS RBCs may also contribute to an increased adhesive response to adenylate cyclase activation compared to normal RBCs. In addition, we have obtained the first evidence that ERK1/2 is conserved in enucleated human erythrocytes and that ERK1 is recruited to bind to SS RBC membranes as a result of adenylate cyclase activation, suggesting that it might be involved in regulating cell adhesion.