Phosphorylation site specificities of glycogen synthase kinases: determination by peptide mapping using high-performance liquid chromatography.
A method is described which separates the various phosphorylation sites in glycogen synthase based on reverse phase high-performance liquid chromatography (HPLC) of tryptic 32P-peptides. Using this method we studied the phosphorylation site specificities of the kinases which act on glycogen synthase. The cAMP-dependent protein kinase phosphorylated sites 1a, 1b, and 2, whereas casein kinase II phosphorylated only site 5. Two calcium, calmodulin-dependent kinases, phosphorylase kinase and liver calmodulin-dependent synthase kinase, both phosphorylated site 2, and the latter enzyme also phosphorylated site 1b. A cAMP-independent kinase (kinase 4) purified from liver also specifically phosphorylated site 2. Synthase kinase 3 catalyzed the phosphorylation of only site 3. This HPLC method was also used to establish that all of these sites were subject to phosphorylation in vivo.
D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D014357:Trypsin
D010446:Peptide Fragments; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin