Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates.
Recognition of substrates by the protein kinase glycogen synthase kinase 3 (GSK-3) usually requires prior phosphorylation of the substrate. Using a peptide based on the glycogen synthase sequence PRPAS(3a)VPPS (3b)PSLS(3c)RHSS(4)PHQS(5)EDEEEP (where the numbers in parentheses denote sites of phosphorylation), we showed previously that phosphorylation of site 5 by casein kinase II was necessary for GSK-3 to phosphorylate the peptide at sites 3a, 3b, 3c, and 4 (Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048). In the present study, variant peptides were synthesized in which sites 3a, 3b, 3c, and 4 were individually replaced by Ala residues (denoted Ala-3c, etc.). All of the variant peptides were substrates for casein kinase II. The peptide Ala-4,Ser(P)-5 was not a substrate for GSK-3 confirming the minimal recognition sequence for the protein kinase as -SXXXS(P)-. The peptides Ala-3c,Ser(P)-5, Ala-3b,Ser(P)-5, and Ala-3a,Ser(P)-5, however, were all good substrates for GSK-3 with apparent Km values in the range 3-6 microns, comparable with that of the parent peptide. GSK-3 could introduce 1, 2, and 3 phosphates, respectively, into these substrates, always COOH-terminal to the substituted Ala residue. Ala-4,Ser(P)-5 and Ala-3c,Ser(P)-4,Ser(P)-5 were competitive inhibitors for phosphorylation of the parent peptide, with Ki values of 2 and 5 microns, respectively. The data suggest (i) that GSK-3 recognizes serines in the motif -SXXXS(P)-, and (ii) that multiple phosphorylation of the peptide substrate has an obligate order, with the sequential formation of new recognition sequences.
Fiol
C J
CJ
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
Wang
A
A
Roeske
R W
RW
Roach
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038341:Glycogen Synthase Kinases; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis*; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D014357:Trypsin
D010455:Peptides; D010748:Phosphopeptides; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin