Atypical Activation of Plasma Membrane-Bound ERK1/2 Is Associated with Regulation of Sickle Red Cell Adhesion to Endothelium

Abstract 266

Abnormal adhesion of erythrocytes homozygous for hemoglobin S (SS RBCs) and vaso-occlusion are hallmarks of sickle cell disease (SCD). SS RBC adhesion can be activated via stimulation of the cAMP/PKA pathway by the stress hormone epinephrine. We have found that the extracellular signal-regulated kinase-1 and 2 (ERK1/2) is abundantly expressed in mature RBCs and is bound to the isolated plasma membrane. Because aberrations in ERK1/2 signaling are associated with various pathologies, we predicted that this prototypical molecular regulator of cell division and differentiation remained functional in terminally differentiated SS RBCs and plays a role in regulating RBC adherence. Western blot analysis of RBC ghosts showed that ERK1/2 was phosphorylated to some degree in SS but not normal RBCs. Although this basal ERK1/2 phosphorylation was not detected in all SS RBC samples tested, ERK1/2 underwent increased phosphorylation (p<0.01) in all samples tested (n=8) in response to epinephrine stimulation. Incubation of SS RBCs with the MEK inhibitor (MEKI) U0126, which specifically inhibits MEK1/2, the upstream kinase of ERK activation, completely abolished epinephrine-induced ERK1/2 phosphorylation. In contrast, epinephrine completely failed to activate ERK1/2 in normal RBCs. We further confirmed that ERK1/2 preserved its activity in SS RBCs by determining that ERK1/2 immunoprecipitated from sham-treated SS RBCs was able to phosphorylate to some extent myelin basic protein (MBP), the ERK specific substrate. However, MBP phosphorylation by ERK1/2 isolated from epinephrine-treated SS RBCs increased by 62% compared to MPB phosphorylation by ERK1/2 isolated from sham-treated cells (n=4, p<0.03). This indicates that ERK1/2 is active in SS RBCs and that epinephrine amplifies its activity. Active ERK1/2 was also found to act downstream of cAMP/PKA, since treatment of SS RBCs with forskolin, which directly activates adenylyl cyclase to produce cAMP, increased ERK1/2 phosphorylation, and the PKA-specific inhibitor 14–22 amide completely blocked the effect of epinephrine on ERK phosphorylation (n=3, p<0.001). Importantly, we found that activation of ERK1/2 signaling was implicated in adhesion of SS RBC to endothelial cells (ECs). Epinephrine significantly up-regulated SS RBC adhesion at a shear stress of 2 dynes/cm² (p < 0.001). However, SS RBC adhesion induced by epinephrine was completely inhibited by pre-treatment of SS RBCs with MEKI U0126 (p < 0.001). PhosphorImager analysis of immunoprecipitated ³²P-radiolabeled ICAM-4, which mediates SS RBC adhesion to ECs, showed that the previously described phosphorylation of ICAM-4 in response to epinephrine was dependent on PKA and MEK1/2/ERK1/2. Furthermore, addition of recombinant ERK2 to RBC ghosts, followed by mass spectrometric analysis, showed that ERK phosphorylated its consensus motif on adducins α and β and dematin; band 4.1 also underwent phosphorylation but not at an ERK consensus motif. This suggests that phosphorylation of cytoskeletal proteins may induce membrane protein conformational changes that render ICAM-4 accessible to phosphorylation by an as yet unidentified kinase. Finally, SS RBC adhesion was closely related to the onset of ERK1/2 activation. Within 1 minute of stimulation, epinephrine induced a significant increase in both SS RBC adhesion to ECs and phosphorylation of ERK1/2 (p < 0.001). Both SS RBC adhesion and ERK phosphorylation decreased after longer exposure to epinephrine (30 min vs 1 min, p < 0.001 for each). Addition of recombinant ERK2 to SS RBC ghosts, followed by mass spectrometry, also showed that phosphorylation of the ERK consensus motif on adenylyl cyclase-associated protein 1 increased 5-fold (p < 0.0003). These data suggest that activation of adenylyl cyclase-associated protein 1, which is known to inhibit adenylyl cyclase activity, may negatively regulate activation of ERK in SS RBCs. In summary, our data indicate that ERK1/2 activity is atypically preserved in SS RBCs. These data also suggest that ICAM-4 adhesive function is regulated by ERK activation, and that ERK activity could probably be turned off by ERK-induced inactivation of adenylyl cyclase. Activation of ERK1/2 in SS RBCs is an extremely interesting phenomenon in SCD physiopathology, since its mode of action could become a potential therapeutic target, and MEK inhibitors are currently in development as therapeutic agents in cancer.