Crystal Structure of Glycogen Synthase Kinase 3β Structural Basis for Phosphate-Primed Substrate Specificity and Autoinhibition

Glycogen synthase kinase 3 beta (GSK3 beta) plays a key role in insulin and Wnt signaling, phosphorylating downstream targets by default, and becoming inhibited following the extracellular signaling event. The crystal structure of human GSK3 beta shows a catalytically active conformation in the absence of activation-segment phosphorylation, with the sulphonate of a buffer molecule bridging the activation-segment and N-terminal domain in the same way as the phosphate group of the activation-segment phospho-Ser/Thr in other kinases. The location of this oxyanion binding site in the substrate binding cleft indicates direct coupling of P+4 phosphate-primed substrate binding and catalytic activation, explains the ability of GSK3 beta to processively hyperphosphorylate substrates with Ser/Thr pentad-repeats, and suggests a mechanism for autoinhibition in which the phosphorylated N terminus binds as a competitive pseudosubstrate with phospho-Ser 9 occupying the P+4 site.

[1]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[2]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[3]  Paul Polakis,et al.  The oncogenic activation of β-catenin , 1999 .

[4]  E. Goldsmith,et al.  Dimerization in MAP-kinase signaling. , 2000, Trends in biochemical sciences.

[5]  Jörg Stappert,et al.  β‐catenin is a target for the ubiquitin–proteasome pathway , 1997 .

[6]  Akira Kikuchi,et al.  Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK‐3β and β‐catenin and promotes GSK‐3β‐dependent phosphorylation of β‐catenin , 1998 .

[7]  John Kuriyan,et al.  Crystal structure of the Src family tyrosine kinase Hck , 1997, Nature.

[8]  P. Roach,et al.  Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a). , 1993, The Journal of biological chemistry.

[9]  T. Dale,et al.  An assay for glycogen synthase kinase 3 (GSK-3) for use in crude cell extracts. , 1998, Analytical biochemistry.

[10]  P. O'Connor,et al.  The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation. , 2001, Cell.

[11]  S. Hubbard,et al.  Crystal structure of the tyrosine kinase domain of the human insulin receptor , 1994, Nature.

[12]  P. Cohen,et al.  Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B , 1995, Nature.

[13]  Michael J. Eck,et al.  Three-dimensional structure of the tyrosine kinase c-Src , 1997, Nature.

[14]  Elizabeth J. Goldsmith,et al.  Activation Mechanism of the MAP Kinase ERK2 by Dual Phosphorylation , 1997, Cell.

[15]  D. M. Ferkey,et al.  GBP, an Inhibitor of GSK-3, Is Implicated in Xenopus Development and Oncogenesis , 1998, Cell.

[16]  L. Johnson,et al.  Expression of the phosphorylase kinase γ subunit catalytic domain in Escherichia coli , 1992 .

[17]  L. Johnson,et al.  Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product. , 1995, Structure.

[18]  A. Sparks,et al.  Identification of c-MYC as a target of the APC pathway. , 1998, Science.

[19]  A. Kikuchi,et al.  Tyrosine dephosphorylation of glycogen synthase kinase‐3 is involved in its extracellular signal‐dependent inactivation , 1996, FEBS letters.

[20]  O. MacDougald,et al.  Glycogen Synthase Kinase 3 Is an Insulin-Regulated C/EBPα Kinase , 1999, Molecular and Cellular Biology.

[21]  James R. Woodgett,et al.  Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signalling in intact cells , 1996, Current Biology.

[22]  A. Depaoli-Roach,et al.  Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1. , 1988, Archives of biochemistry and biophysics.

[23]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[24]  J. Woodgett,et al.  Glycogen synthase kinase-3: functions in oncogenesis and development. , 1992, Biochimica et biophysica acta.

[25]  P. Polakis.  Wnt signaling and cancer. , 2000, Genes & development.

[26]  L. Johnson,et al.  The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition , 1997, The EMBO journal.

[27]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[28]  A. Kimmel,et al.  The Novel Tyrosine Kinase ZAK1 Activates GSK3 to Direct Cell Fate Specification , 1999, Cell.

[29]  Margaret Robertson,et al.  Identification and characterization of the familial adenomatous polyposis coli gene , 1991, Cell.

[30]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[31]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[32]  Elizabeth J. Goldsmith,et al.  Atomic structure of the MAP kinase ERK2 at 2.3 Å resolution , 1994, Nature.

[33]  P. Roach,et al.  Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates. , 1990, The Journal of biological chemistry.

[34]  Sung-Hou Kim,et al.  Crystal structure of cyclin-dependent kinase 2 , 1993, Nature.

[35]  H. Usui,et al.  GSK-3β-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by β-catenin and protein phosphatase 2A complexed with Axin , 2000, Oncogene.

[36]  E. Goldsmith,et al.  Activity of the MAP kinase ERK2 is controlled by a flexible surface loop. , 1995, Structure.

[37]  P. Cohen,et al.  A GSK3‐binding peptide from FRAT1 selectively inhibits the GSK3‐catalysed phosphorylation of Axin and β‐catenin , 1999, FEBS letters.

[38]  D. M. Ferkey,et al.  Interaction among Gsk-3, Gbp, Axin, and APC in Xenopus Axis Specification , 2000, The Journal of cell biology.

[39]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[40]  P. Graves,et al.  Phosphate groups as substrate determinants for casein kinase I action. , 1990, The Journal of biological chemistry.

[41]  M. Roussel,et al.  Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. , 1998, Genes & development.

[42]  J. Woodgett,et al.  Modulation of the glycogen synthase kinase‐3 family by tyrosine phosphorylation. , 1993, The EMBO journal.

[43]  K. Longenecker,et al.  Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition. , 1996, Journal of molecular biology.

[44]  P. Jeffrey,et al.  Structural basis of cyclin-dependent kinase activation by phosphorylation , 1996, Nature Structural Biology.

[45]  W. Hackmann.  Tesla's sparks of imagination , 1993, Nature.

[46]  S. Altschul,et al.  Identification of FAP locus genes from chromosome 5q21. , 1991, Science.

[47]  B. Neel,et al.  Specific modulation of ectodermal cell fates in Xenopus embryos by glycogen synthase kinase. , 1995, Development.

[48]  P. Cohen,et al.  The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction. , 1999, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[49]  A. Harwood,et al.  Lithium therapy and signal transduction. , 2000, Trends in pharmacological sciences.

[50]  M. Cobb,et al.  Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. , 1993, The Journal of biological chemistry.

[51]  Paul Polakis,et al.  Downregulation of β-catenin by human Axin and its association with the APC tumor suppressor, β-catenin and GSK3β , 1998, Current Biology.

[52]  L. Johnson,et al.  Active and Inactive Protein Kinases: Structural Basis for Regulation , 1996, Cell.

[53]  R M Sweet,et al.  Crystal structure of casein kinase‐1, a phosphate‐directed protein kinase. , 1995, The EMBO journal.

[54]  J Mao,et al.  Axin and Frat1 interact with Dvl and GSK, bridging Dvl to GSK in Wnt‐mediated regulation of LEF‐1 , 1999, The EMBO journal.