Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1 |
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Affiliation: | 1. Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100083, China;2. Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Peking University, Beijing 100083, China;3. Feihe Research Institute, Heilongjiang Feihe Dairy Co., Ltd, Beijing 100016, China;4. PKUHSC-China Feihe Joint Research Institute of Nutrition and Healthy Lifespan Development, Beijing 100083, China;5. National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China;6. Institute of Food Science and Technology, Chinese Academy of Agricultural Science, Beijing 100081, China |
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Abstract: | Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When 32P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1. |
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