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.
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.
Fiol
C J
CJ
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
Mahrenholz
A M
AM
Wang
Y
Y
Roeske
R W
RW
Roach
P J
PJ
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
D010455:Peptides; D010748:Phosphopeptides; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D047388:Casein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin