The stereospecificity of protein kinases

To test whether cellular protein kinases exist that phosphorylate D-amino acid residues, a method was developed for separating O-phospho-D-serine from O-phospho-L-serine and O-phospho-L-tyrosine from O-phospho-D-tyrosine. This was accomplished by converting these amino acids to the L-leucyl dipeptide derivatives followed by separation of the diastereomers by anion-exchange high-performance liquid chromatography. The enantiomeric content of these D- and L-residues were measured in hydrolysates of 32P-labeled proteins produced by the protein kinases of human erythrocytes and the tyrosyl protein kinase of the Abelson leukemia virus. We found no measurable D-phosphoserine in erythrocyte membrane proteins under conditions where a 1% content of this residue relative to L-phosphoserine would have been detected. These values can be used to place an upper hypothetical limit on the fraction of erythrocyte protein kinase activity that is specific for serine residues in the D-configuration. In separate experiments, we examined the specificity of the tyrosyl protein kinases. We found that all of the phosphotyrosine that we isolated from the erythrocyte band 3 NH2-terminal fragment and from the autophosphorylation of the Abelson virus tyrosyl kinase was in the L-configuration.