Phosphorylation of proteasomes in mammalian cells. Identification of two phosphorylated subunits and the effect of phosphorylation on activity.
The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracellular protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated 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 [32P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome-specific polyclonal antibodies. Subsequent analysis by two-dimensional 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 identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cGMP-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 dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel-filtration from those complexed with regulatory complexes to form the 26S proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26S proteinase than in free proteasomes.
Mason
G G
GG
Department of Biochemistry, University of Leicester, UK.
Hendil
K B
KB
Rivett
A J
AJ
D000135:Acid Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D051381:Rats
D000906:Antibodies; D009097:Multienzyme Complexes; D010748:Phosphopeptides; D010768:Phosphoserine; D000135:Acid Phosphatase; D010450:Endopeptidases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease