The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation.
BACKGROUND
Glycogen synthase kinase-3 (GSK-3) sequentially phosphorylates four serine residues on glycogen synthase (GS), in the sequence SxxxSxxxSxxx-SxxxS(p), by recognizing and phosphorylating the first serine in the sequence motif SxxxS(P) (where S(p) represents a phosphoserine). FRATtide (a peptide derived from a GSK-3 binding protein) binds to GSK-3 and blocks GSK-3 from interacting with Axin. This inhibits the Axin-dependent phosphorylation of beta-catenin by GSK-3.


RESULTS
Structures of uncomplexed Tyr216 phosphorylated GSK-3beta and of its complex with a peptide and a sulfate ion both show the activation loop adopting a conformation similar to that in the phosphorylated and active forms of the related kinases CDK2 and ERK2. The sulfate ion, adjacent to Val214 on the activation loop, represents the binding site for the phosphoserine residue on 'primed' substrates. The peptide FRATtide forms a helix-turn-helix motif in binding to the C-terminal lobe of the kinase domain; the FRATtide binding site is close to, but does not obstruct, the substrate binding channel of GSK-3. FRATtide (and FRAT1) does not inhibit the activity of GSK-3 toward GS.


CONCLUSIONS
The Axin binding site on GSK-3 presumably overlaps with that for FRATtide; its proximity to the active site explains how Axin may act as a scaffold protein promoting beta-catenin phosphorylation. Tyrosine 216 phosphorylation can induce an active conformation in the activation loop. Pre-phosphorylated substrate peptides can be modeled into the active site of the enzyme, with the P1 residue occupying a pocket partially formed by phosphotyrosine 216 and the P4 phosphoserine occupying the 'primed' binding site.
Bax
B
B
Department of Structural Biology, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AD, United Kingdom. benjamin_d_bax@gsk.com
Carter
P S
PS
Lewis
C
C
Guy
A R
AR
Bridges
A
A
Tanner
R
R
Pettman
G
G
Mannix
C
C
Culbert
A A
AA
Brown
M J
MJ
Smith
D G
DG
Reith
A D
AD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D060466:Axin Protein; D001665:Binding Sites; D001667:Binding, Competitive; D042846:CDC2-CDC28 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D002460:Cell Line; D018360:Crystallography, X-Ray; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D015534:Trans-Activators; D051176:beta Catenin
D060466:Axin Protein; D003598:Cytoskeletal Proteins; D008024:Ligands; D010455:Peptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D051176:beta Catenin; D012694:Serine; D038341:Glycogen Synthase Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019950:Mitogen-Activated Protein Kinase 1; D038362:Glycogen Synthase Kinase 3