Phosphorylation of ATPase subunits of the 26S proteasome.
The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.
Mason
G G
GG
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.
Murray
R Z
RZ
Pappin
D
D
Rivett
A J
AJ
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002460:Cell Line; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008099:Liver / Q000201:enzymology; D008168:Lung; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry*; D010766:Phosphorylation; D011233:Precipitin Tests / Q000379:methods; D046988:Proteasome Endopeptidase Complex; D051381:Rats
D000911:Antibodies, Monoclonal; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases