Serine, threonine and tyrosine (STY) are the 3 commonly studied phosphorylated amino acids. But in reality, there are 9 amino acids capable of being phosphorylated including histidine. Their study has been limited by a lack of technical resources. Since 2015, new protocols and tools have been developed for their study. Initial exploratory studies have shown an important role for these phosphorylations. Double KO of NME1 and NME2, the only known histidine kinases, in mice resulted in perinatal death associated with anemia and iron deficiency, although the molecular mechanisms involved were not elucidated. Previous study showed that in red blood cells, histidine kinases are among the three most highly expressed kinases, although their role in these cells remains unknown.



In order to investigate the involvement of histidine phosphorylations in erythropoiesis and red blood cells, we first set up two mass spectrometry protocols for the analysis of these phosphorylations, based on published protocols. The selected protocol allowed us to yield more than 250 histidine phosphorylations on average, compared with an average of 33 phosphorylations for conventional phosphorylation study protocols, in erythroid UT7EPO.



Analysis of the functional data revealed an enrichment of zinc-finger proteins for histidine-phosphorylated proteins compared with the total proteome of the line, including BCL11A, a key erythroid regulator for fetal to adult hemoglobin switch. This phosphorylation was validated by immunoprecipitation and western blot. The invalidation by CRISPR Cas9 of NME1/2 in a erythroid cell line led to the re-expression of the fetal hemoglobin suggesting the implication of NME1/2 in hemoglobin switch possibly via BCL11A histidine phosphorylation.



The exploration by mass spectrometry led to the identification of a new regulatory system for fetal to adult hemoglobin switch. Further investigations are needed to confirm histidine phosphorylation involvement in this regulation.