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Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates
Ist Teil von
The Journal of biological chemistry, 1990-04, Vol.265 (11), p.6061-6065
Ort / Verlag
Bethesda, MD: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
1990
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
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.