Differential Kinase Activity in Aged Erythrocytes Following Nutrient Replenishment: Insights from Phosphoproteomics and Motif Analysis

The erythrocyte is unique, lacking a nucleus, mitochondria, and other components that orchestrate cell behavior. It is subjected to some of the most extreme and varied environments in the body. Without the mitochondria and nucleus, a key to method to alter behavior in response to these changes is protein kinase activity and protein phosphorylation. As phosphorylation utilizes a valuable resource to the red cell, energy in the form of adenosine-triphosphate (ATP), we reason that this process would be regulated to avoid unnecessary energetic waste. By comparing stored, aged, donor erythrocytes to those in nutrient-rich media, we have performed phosphoproteomics to study protein phosphorylation in erythrocytes. Using motif analysis, these phosphorylation events have been mapped to the most likely kinase performing the phosphorylation and provide insights about the signaling pathways active within the red cell.

Leukoreduced and irradiated de-identified erythrocytes stored in adenine saline solution were acquired from the Brigham and Women's Transfusion Service. Cells were washed in cold phosphate buffer at 4 degrees using centrifugation. Erythrocytes were lysed and hemoglobin was depleted using nickel-nitrilotriacetic columns to optimize for detection of low abundance proteins. Quantitative phosphoproteomics using high-resolution mass spectrometry with isobaric tandem mass tag labeling was performed on 8 donor erythrocyte units before and after 3-hour incubation in nutrient-rich media at 37 degrees.

Our study identified 2,231 proteins and 3,232 phosphorylated sites. Analysis using paired t-tests for multiple comparisons revealed 655 significant differentially phosphorylated peptides of 401 distinct proteins. Pathway analysis linked these to protein metabolism, stress response, regulation of apoptosis, energy metabolism, and calcium signaling among others. Using published kinase specificity predictions, the differentially phosphorylated sites were used to computationally predict which kinases were activated or suppressed in response to nutrient incubation. Among the kinases predicted to display lower activity in stored vs. nutrient treated erythrocytes are the p38 mitogen-activated protein kinases (MAPK), extra-cellular signal regulated kinase (ERK), and cyclin-dependent kinases (CDK). Kinases with higher activity in stored conditions include p21-activated kinases, calcium/calmodulin-dependent protein kinase (CAMK), and AMP-activated protein kinase (AMPK). AMPK was of particular interest due to its role in responding to energetic stress; however its signaling and downstream phosphorylation in erythrocytes is poorly understood. Analysis of phosphorylation motifs revealed 99 sites targeted by the AMPK consensus motif; of these, 68 decrease in response to media whereas 31 increase. Downstream of AMPK, a reduction in Raptor phosphorylation at serine 722 (RBC = 7.281 vs. Media = 5.219; q = 0.0188) was observed following media treatment along with reductions in AMPKalpha1 phosphorylation at threonine 172 (RBC = 6.749 vs. Media = 5.751; q = 0.0689). This suggests that the stored donor units display elevated AMPK activity, consistent with energetic stress, when compared with those in nutrient rich conditions. To investigate this, erythrocytes were treated with kinase modulators focused on the AMPK and p21-activated kinase (PAK) pathways, with hemolysis monitored using free hemoglobin in the supernatant of centrifuged samples. Inhibition of PAK with IPA-3 and activation of AMPK with high doses of AICAR both resulted in increased hemolysis. The targets and mechanisms of erythrocyte destruction following these treatments are currently under investigation.

This work provides insight into the phosphorylation events and kinase activity during red cell storage and on the transition in nutrient-rich conditions as would be expected following transfusion. Work is underway to elucidate the targets of these pathways using kinase inhibition and activation as well as genetic deletion and mutation to better understand how erythrocytes respond to their environment and cope with diverse stressors. By focusing on kinase signaling in erythrocytes, we aim expand our knowledge of the critical pathways active in red cells, identify key phosphorylation events that modulate cell responses, and optimize storage of red cell donor units.

Cantley:Novartis Pharmaceuticals, Scorpion, Larkspur: Consultancy; Petra Pharmaceuticals, Faeth Therapeutics: Current equity holder in private company.