Identification of novel glycogen synthase kinase-3beta substrate-interacting residues suggests a common mechanism for substrate recognition.
Substrate recognition and specificity are essential for the reliability and fidelity of protein kinase function. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates. However, how the enzyme selects its phosphorylated substrates is unknown. Here, we combined in silico modeling with mutagenesis and biological studies to identify GSK-3-substrate interaction sites located within its binding cleft. Protein-protein docking of GSK-3beta and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe67, Gln89, and Asn95 of GSK-3beta as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3beta phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3beta mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and beta-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wild-type GSK-3beta. Our studies provide important additional understanding of how GSK-3beta recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3beta residues: Phe67, Gln89, and Asn95, which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity.
Ilouz
Ronit
R
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel.
Kowalsman
Noga
N
Eisenstein
Miriam
M
Eldar-Finkelman
Hagit
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D051176:beta Catenin / Q000378:metabolism
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3