PHORBOL 12-MYRISTATE 13-ACETATE-INDUCED PHOSPHORYLATION OF OP18 IN JURKAT T-CELLS - IDENTIFICATION OF PHOSPHORYLATION SITES BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY

Op18 is a widely expressed, cell cycle-regulated, phosphoprotein invol ved in signal transduction of a variety of stimuli. In actively prolif erating Jurkat T cells which express Op18 at high level, phorbol 12-my ristate 13-acetate (PMA) treatment induces a rapid increase in the lev el of several Op18 phosphorylated forms. To determine phosphorylation sites involved in the PMA effect, the major Op18 phosphorylated forms were resolved in Jurkat T cells, before and after treatment with PMA, using preparative immobilized pH gradient-based two-dimensional polyac rylamide gel electrophoresis. Tryptic fragments of phosphorylated Op18 were analyzed by two-dimensional thin layer peptide mapping and were resolved by reverse-phase high performance liquid chromatography prior to analysis by matrix-assisted laser desorption ionization mass spect rometry. Phosphorylation sites were identified by further treatment of the proteolytic fragments with different enzymes and determination of the mass shifts by matrix-assisted laser desorption ionization mass s pectrometry. Two major phosphorylation sites were identified. Low cons titutive levels of phosphorylation at Ser25 and Ser38 in Op18a and Op1 8b was demonstrated. Treatment with PMA resulted in enhanced phosphory lation of Ser25 in Op18a and of both Ser25 and Ser38 in Op18b. Taken t ogether with prior studies of Op18 phosphorylation, the data suggest t hat Op18 phosphorylation occurs at identical sites in different tissue s and organisms.