Histidine phosphorylation in histones and in other mammalian proteins.

The investigation of protein histidine phosphorylation has required the development of a number of methods that differ from traditional methods of phosphoprotein analysis that were developed to study phosphorylation of serine, threonine, and tyrosine, which are, unlike phosphohistidine, acid-stable. The investigation of histidine phosphorylation is further complicated by the fact that in mammalian proteins, phosphorylation appears to occur at either 1-N or 3-N positions of the imidazole ring, depending on the source of the kinase. In this review, we describe methods developed for phosphoamino acid analysis to detect phosphohistidine, including the determination of the isoform present, using chromatographic and mass spectrometric analysis of phosphoprotein hydrolysates and 1H- and 31P NMR analysis of intact phosphoproteins and phosphopeptides. We also describe methods for the assay of protein histidine kinase activity, including a quantitative assay of alkali-stable, acid-labile protein phosphorylation, and an in-gel kinase assay applied to histidine kinases. Most of the detailed descriptions of methods are as they are applied in our laboratory to the investigation of histone H4 phosphorylation and histone H4 histidine kinases, but which can be applied to the phosphorylation of any proteins and to any such histidine kinases.