Protein Kinase C-α Produces Reciprocal Effects on the Phorbol Ester Stimulated Tyrosine Phosphorylation of a 50 kDa Kinase in Jurkat Cells

Abstract

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly-(Glu⁴,Tyr¹). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the α-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the α-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with ³²P_i indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein under in vivo conditions.

These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5′-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by α-PKC.