title: Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3.
abstract: The mechanism for synergistic phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase II was studied using a synthetic peptide which contains the sequence of a potentially important proline/serine-rich regulatory region of rabbit muscle glycogen synthase. The peptide, Ac-PRPAS(3a)VPPS(3b)PSLS(3c)RHSS(4)PHQS(5) EDEEEP-amide, has five known phosphorylation sites of the native enzyme designated sites 3a, 3b, 3c, 4, and 5, which are spaced every fourth residue. The peptide was phosphorylated specifically at site 5 by casein kinase II with an apparent Km of 23 microM, but it was not phosphorylated by GSK-3. However, after initial phosphorylation of site 5 by casein kinase II, the peptide became an effective substrate for GSK-3 with an apparent Km of 2 microM. GSK-3 introduced up to four phosphates and appeared to catalyze the sequential modification of sites 4, 3c, 3b, and 3a, respectively. The results can be explained if GSK-3 recognizes the sequence -SXXXS(P). Phosphorylation of site 5 by casein kinase II creates this recognition site. Thereafter, each successive phosphorylation introduced by GSK-3 generates a new recognition site. The results provide a molecular basis to explain the synergistic action of casein kinase II and GSK-3 that is also observed with native glycogen synthase. In addition, this investigation emphasizes how protein recognition sites in some cellular targets may have to be formed post-translationally.
authors.affiliation: Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
authors.forename: C J
authors.initials: CJ
authors.lastname: Fiol
authors.forename: A M
authors.initials: AM
authors.lastname: Mahrenholz
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: R W
authors.initials: RW
authors.lastname: Roeske
authors.forename: P J
authors.initials: PJ
authors.lastname: Roach
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047388:Casein Kinases; D038341:Glycogen Synthase Kinases; D007525:Isoelectric Focusing; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010455:Peptides / Q000138:chemical synthesis; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D014357:Trypsin
chemical_list: D010455:Peptides; D010748:Phosphopeptides; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D047388:Casein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin