PHOSPHORYLATION OF PROTEASOMES IN MAMMALIAN-CELLS - IDENTIFICATION OF2 PHOSPHORYLATED SUBUNITS AND THE EFFECT OF PHOSPHORYLATION ON ACTIVITY

The proteasome, a multimeric protease, plays an important role in nonl ysosomal pathways of intracellular protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are p hosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat-1 fibroblasts were grown in the presence of [P-32]phos phate and proteasomes were immunoprecipitated from cell lysates with p roteasome-specific polyclonal antibodies. Subsequent analysis by two-d imensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L-132), under i dentical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in bo th C8 and C9. Examination of the sequence of C9 showed a potential cCM P-dependent phosphorylation site (-Arg3-Arg-Tyr-Asp-Ser-Arg8-), whilst C8 contains several potential casein kinase II phosphorylation sites, Following immunoprecipitation by a monoclonal antibody and dephosphor ylation by acid phosphatase, proteasomes were observed to have signifi cantly lower activities when compared to phosphorylated proteasomes, i mplying that phosphorylation may be an important mechanism of regulati ng proteasome function. Free proteasomes were separated by gel-filtrat ion from those complexed with regulatory complexes to form the 26S pro teinase. The ratio of phosphorylation of C8 and C9 was found to be ver y similar in the two complexes but the level of phosphorylation was hi gher in the 26S proteinase than in free proteasomes.