期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 431, 期 1, 页码 9-15出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2004.07.020
关键词
proteasome; 20S proteasome; alpha 7; phosphorylation; mass spectrometry; yeast; casein kinase II; identification; modification; post-translational modification
The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively. (C) 2004 Elsevier Inc. All rights reserved.
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