GSK-3: tricks of the trade for a multi-tasking kinase

Abstract

Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine kinase found in all eukaryotes. The enzyme is a key regulator of numerous signalling pathways, including cellular responses to Wnt, receptor tyrosine kinases and G-protein-coupled receptors and is involved in a wide range of cellular processes, ranging from glycogen metabolism to cell cycle regulation and proliferation. GSK-3 is unusual in that it is normally active in cells and is primarily regulated through inhibition of its activity. Another peculiarity compared with other protein kinases is its preference for primed substrates, that is, substrates previously phosphorylated by another kinase. Several recent advances have improved our understanding of GSK-3 regulation in multiple pathways. These include the solution of the crystal structure of GSK-3, which has provided insight into GSK-3's penchant for primed substrates and the regulation of GSK-3 by serine phosphorylation, and findings related to the involvement of GSK-3 in the Wnt/beta-catenin and Hedgehog pathways. Finally, since increased GSK-3 activity may be linked to pathology in diseases such as Alzheimer's disease and non-insulin-dependent diabetes mellitus, several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture experiments, these new inhibitors hold promise as therapeutic agents.

Fig. 1

Schematic representation of mammalian GSK-3α and GSK-3β. Sites of serine and tyrosine phosphorylation are indicated with blue arrowheads. The glycine-rich amino-terminal domain unique to GSK-3α and the conserved kinase domain shared by both isoforms are highlighted.

Fig. 2

Fig. 2A. Regulation of GSK-3β activity by serine phosphorylation. In the resting cell, GSK-3β is constitutively active. Both un-primed substrates and substrates phosphorylated by a priming kinase (PK) are capable of being phosphorylated by the active GSK-3β. The priming phospho-residue at position N + 4, binds a pocket of positive charge arising from the arginine (R) and lysine (K) residues indicated. This directs a serine or threonine at position N to the active catalytic site (c.s.). When an inactivating kinase (IK) such as PKB/Akt phosphorylates GSK-3β on serine 9 (S9), the phosphorylated amino-terminus becomes a primed pseudo-substrate that occupies the positive binding pocket and active site of the enzyme, acting as a competitive inhibitor for true substrates. This prevents phosphorylation of any substrates. 2B. Effect of mutating arginine 96 to alanine (R96A) on GSK-3β activity. Since R96 is a crucial component of the positive pocket that binds primed substrates, its mutation to an uncharged ala residue disrupts the pocket so that primed substrates can no longer bind. The enzyme retains activity. Also, the S9-phosphorylated pseudosubstrate is no longer capable of inactivating the enzyme. As a consequence, GSK-3β whether S9-phosphorylated or not, can phosphorylate unprimed substrates, but not primed substrates.

Fig. 2

Fig. 2A. Regulation of GSK-3β activity by serine phosphorylation. In the resting cell, GSK-3β is constitutively active. Both un-primed substrates and substrates phosphorylated by a priming kinase (PK) are capable of being phosphorylated by the active GSK-3β. The priming phospho-residue at position N + 4, binds a pocket of positive charge arising from the arginine (R) and lysine (K) residues indicated. This directs a serine or threonine at position N to the active catalytic site (c.s.). When an inactivating kinase (IK) such as PKB/Akt phosphorylates GSK-3β on serine 9 (S9), the phosphorylated amino-terminus becomes a primed pseudo-substrate that occupies the positive binding pocket and active site of the enzyme, acting as a competitive inhibitor for true substrates. This prevents phosphorylation of any substrates. 2B. Effect of mutating arginine 96 to alanine (R96A) on GSK-3β activity. Since R96 is a crucial component of the positive pocket that binds primed substrates, its mutation to an uncharged ala residue disrupts the pocket so that primed substrates can no longer bind. The enzyme retains activity. Also, the S9-phosphorylated pseudosubstrate is no longer capable of inactivating the enzyme. As a consequence, GSK-3β whether S9-phosphorylated or not, can phosphorylate unprimed substrates, but not primed substrates.

Fig. 3

Central Role of GSK-3 in the Wnt/β-catenin Pathway. In unstimulated cells, phosphorylation by CKI (S45) primes β-catenin for subsequent phosphorylation by GSK-3 (S41,S37,S33) which targets β-catenin for ubiquitination and proteasomal degradation. The ankyrin repeat protein, Diversin (Div) may help recruit CKI to the destruction complex. Wnt stimulation activates the receptor Frizzled, which then signals through Dishevelled (Dvl), using an unclear mechanism, to inactivate β-catenin phosphorylation. Unphosphorylated β-catenin accumulates and then translocates to the nucleus where it transactivates genes regulated by TCF/LEF transcription factors. The GSK-3 binding protein (GBP/FRAT) may be involved in transmission of a Wnt signal by regulating GSK-3’s binding to the scaffold protein, Axin.