CD31 (PECAM-1), CDW32 (FC-GAMMA-RII), AND ANTI-HLA CLASS-I MONOCLONAL-ANTIBODIES RECOGNIZE PHOSPHOTYROSINE-CONTAINING PROTEINS ON THE SURFACE OF HUMAN NEUTROPHILS

Although most studies of protein phosphorylation have focused on intra cellular reactions, studies have provided evidence for the existence o f ectoprotein kinase activity on the surface of some cells including h uman neutrophils. The identification and characterization of physiolog ic substrates of ectoprotein kinase activity should aid the understand ing of the role of this enzyme activity in cell function. Immunoprecip itation and subsequent gel electrophoresis of proteins from neutrophil s labeled with [gamma-P-32]ATP under conditions initially designed to detect ectoprotein kinase activity revealed that CD31, CDw32, and anti -HLA class I mAbs specifically recognize phosphoproteins on the surfac e of human neutrophils. Phosphorylation of these proteins was inhibite d by pretreatment of cells with an impermeant sulfhydryl reagent befor e radiolabeling. Phosphoamino acid analysis of the proteins revealed t hat they contained predominantly phosphotyrosine. However, controlled proteolysis of intact cells and purified HLA class I revealed that the HLA class I heavy chain was phosphorylated on the cytoplasmic domain. These results suggest that the molecules recognized by CD31 (PECAM-1) and CDw32 (Fc gamma RII) Abs may also be phosphorylated on cytoplasmi c domains under conditions originally designed to detect ectoprotein k inase activity. Phosphorylation of CD31 (PECAM-1), Fc gamma RII, and H LA class I heavy chain on tyrosine may play a role in regulating their function. These results emphasize that the demonstration that a membr ane protein is an ectokinase substrate is complex and requires the def initive localization of the phosphorylated residue to the extracellula r domain of the protein.