Lithium inhibits GSK-3 kinases, a family of highly conserved serine/threonine protein kinases that have been identified in all eukaryotes examined to date. In vertebrates, GSK-3 is found as two isoforms: GSK-3a and GSK-3^. Lithium has been demonstrated to inhibit both these isoforms in vitro and in vivo through competition for magnesium ion binding (see section 1.2).24,25 GSK-3 phosphor-ylates its amino acid target at an unusual substrate recognition site which contains phosphorylated serine or threonine four residues towards the
Single-letter amino acid sequence for the GSK-3 phosphorylation site on glycogen synthase, defined by single-letter abbreviations. Prior phosphorylation the residue four amino acids towards the 'C'-terminal of the target serine/threonine (S/T) allow the enzyme to recognize the site. The subsequent phosphorylated amino acid may then function to enable further phosphorylation
'C' terminus of the phospho-donor site (Figure 1.1). This acts as a docking site to anchor GSK-3 on its substrate. The resulting phosphorylation can then be used to prime further neighbouring residues. Loss of this priming phosphorylation decreases affinity with the substrate by 100-1000-fold.26 This also means that in most cases GSK-3 works in concert with other kinases.
Glycogen synthase kinase-3 was first identified as an enzyme that phos-phorylates and inactivates glycogen synthase27 in the insulin response pathway (Figure 1.2). In this pathway, insulin turns on GSK-3 activity to inhibit glycogen synthesis. Consequently, in diabetic rat hepatocytes, lithium restores glycogen synthesis in the presence of an overactive insulin pathway. However, GSK-3 has many other targets within the cell including a number of important transcription factors and cytoskeletal proteins.
A significant GSK-3 substrate is the protein ^-catenin, which regulates gene expression by binding to the LEF-1/TCF-3 family of transcription factors.28 GSK-3 phosphorylation of ^-catenin leads to its degradation and hence prevents its accumulation in the nucleus where it binds T-cell factor transcription factors (TCFs) (Figure 1.2). This phosphorylation event is regulated by binding to axin, which brings GSK-3 and a priming kinase, casein kinase 1 (CK 1), in contact with ^-catenin.29 Formation of the axin complex is regulated by the binding of the extra-cellular glycoprotein Wnt to its cell surface receptor Frizzled. In this way Wnt signalling can regulate gene expression through control of ^-catenin degradation. The number of cellular events found to be regulated by Wnt signalling is increasing and includes the control of cell fate during development and cell proliferation. Loss of the GSK-3 phosphorylation sites from ^-catenin, deletions of adenomatous polyposis coli protein (APC), another component of the axin—3-catenin protein complex, and mutations of axin itself are all associated with various forms of human cancer.30 ^-Catenin is also a component of adherens junctions which anchor the actin cytoskele-ton to points of cell-cell contact via cadherin proteins on the cell surface. GSK-3-mediated changes in ^-catenin stability do not affect adherens
Figure 1.2 Glycogen synthase kinase-3 functions in a variety of signalling pathways. Wnt signalling via Frizzled (Frz) and Dishevelled (Dsh) functions to inhibit GSK-3 activity. This effects the phosphorylation and thus stability of the cell cytoskeleton via cell structural components such as microtubule-associated protein MAP 1B and Tau, and the degradation of ,0-catenin which effects transcriptional events in the cell. GSK-3 also functions to modulate apoptotic events within the cell via protein kinase B (PKB) and PI3 kinase signalling, and inhibits glycogen synthase (GS) activity
Figure 1.2 Glycogen synthase kinase-3 functions in a variety of signalling pathways. Wnt signalling via Frizzled (Frz) and Dishevelled (Dsh) functions to inhibit GSK-3 activity. This effects the phosphorylation and thus stability of the cell cytoskeleton via cell structural components such as microtubule-associated protein MAP 1B and Tau, and the degradation of ,0-catenin which effects transcriptional events in the cell. GSK-3 also functions to modulate apoptotic events within the cell via protein kinase B (PKB) and PI3 kinase signalling, and inhibits glycogen synthase (GS) activity junction-mediated cell contact, and cell contact does not alter regulation of ^-catenin-mediated gene expression.
In addition to its role in cell signalling, GSK-3 is also involved in the regulation of the cell cytoskeleton. Inhibition of GSK-3 by lithium, or other GSK-3 inhibitors, affects both microtubule dynamics31 and microtubule polarity.32 These effects are clearly seen in neurons which rely heavily on the microtubule cytoskeleton to grow and maintain their morphology. Lithium treatment causes changes in axonal branching and behaviour of the developing growth cone and thus, the structure of the synapse. Inhibition of GSK-3 in dividing cells also leads to misalignment of the mitotic spindle, affecting chromosome segregation33 and the plane of cell division during development.34 GSK-3 substrates involved in these structural changes include microtubule-associated protein 1B (MAP1B), Tau and APC. Both MAP1B and Tau are microtubule-binding proteins that regulate microtubule dynamics. Tau is hyper-phosphorylated by GSK-3, leading to the generation of the paired helical filaments (PHF) seen in Alzheimer's disease.35 In addition to its association with the axin complex, APC also binds microtubules and when lost leads to chromosomal instability during mitosis.
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