Discrimination between two sites of phosphorylation on adjacent amino acids by phosphorylation site-specific...

A pair of polyclonal antibodies have been produced in rabbits which recognize the two phosphorylated forms of the cardiac muscle protein, phospholamban. The two sites of phosphorylation of this protein are situated on neighboring residues and yet one antibody, PS-16, recognizes the Ser16 phosphoprotein, while the other, PT-17, recognizes the Thr17 phosphoprotein. Neither antibody recognizes phospholamban phosphorylated at "the other site," nor do they recognize free phosphoamino acids or dephosphorylated protein. This represents the most demanding test of the technique of phosphorylation site-specific antibody production to date, which these antibodies have satisfied without ambiguity. These antibodies remain specific for phospholamban in the company of other muscle phosphoproteins and will be invaluable in determining the role of each phosphorylation site in the biology of the heart. They also demonstrate the absolute specificity of phosphorylation site-specific antibodies and augur well for the use of this approach in the study of other phosphoproteins.