GSK3 and its interactions with the PI3K/AKT/mTOR signalling network

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Abstract

Glycogen Synthase Kinase-3 (GSK3 or GSK-3) is a promiscuous protein kinase and its phosphorylation of its diverse substrates has major influences on many areas of physiology and pathology, including cellular metabolism, lineage commitment and neuroscience. GSK3 was one of the first identified substrates of the heavily studied oncogenic kinase AKT, phosphorylation by which inhibits GSK3 activity via the formation of an autoinhibitory pseudosubstrate sequence. This has led to investigation of the role of GSK3 inhibition as a key component of the cellular responses to growth factors and insulin, which stimulate the class I PI 3-Kinases and in turn AKT activity and GSK3 phosphorylation. GSK3 has been shown to phosphorylate several upstream and downstream components of the PI3K/AKT/mTOR signalling network, including AKT itself, RICTOR, TSC1 and 2, PTEN and IRS1 and 2, with the potential to apply feedback control within the network. However, it has been clear for some time that functionally distinct, insulated pools of GSK3 exist which are regulated independently, so that for some GSK3 substrates such as β-catenin, phosphorylation by GSK3 is not controlled by input from PI3K and AKT. Instead, as almost all GSK3 substrates require a priming phosphorylated residue to be 4 amino acids C-terminal to the Ser/Thr phosphorylated by GSK3, the predominant form of regulation of the activity of GSK3 often appears to be through control over these priming events, specific to individual substrates. Therefore, a major role of GSK3 can be viewed as an amplifier of the electrostatic effects on protein function which are caused by these priming phosphorylation events. Here we discuss these different aspects to GSK3 regulation and function, and the functions of GSK3 as it integrates with signalling through the PI3K-AKT-mTOR signalling axis.

Introduction

Reversible phosphorylation is a mechanism of protein regulation which is ubiquitous amongst eukaryotes and almost all human proteins have recognised sites of phosphorylation (Gnad et al., 2011, Hornbeck et al., 2015, Hunter, 2012). Glycogen Synthase Kinase 3 (GSK3) is a serine/threonine directed protein kinase found widely throughout eukaryotes, with over a hundred specifically identified proteins proposed as GSK3 substrates and a much larger number predicted from bioinformatics (Kaidanovich-Beilin and Woodgett, 2011, Linding et al., 2007, Sutherland, 2011). In mammals GSK3 exists in two forms, GSK3α and GSK3β, paralogs which share a highly conserved catalytic domain but differ at both termini and are encoded by separate genes, in humans named GSK3A and GSK3B. Both GSK3α and GSK3β mRNA and protein are routinely detected in most human tissues and although firm quantitation of their concentration is rare, as an example, in human U2OS cells and the murine fibroblast cell line NIH3T3 quantitative proteomics indicated an abundance in the region of 50,000 or 15,000 molecules of GSK3β and around 5000 molecules of GSK3α per cell (Beck et al., 2011, Schwanhausser et al., 2011).

The 500 or so protein kinases encoded in the human genome, often termed the kinome, can be subdivided evolutionarily and functionally into families (Manning et al., 2002). Some kinases show relatively constitutive activity (e.g. CK2) whereas other groups display tightly regulated activity (e.g many members of the Receptor Tyrosine Kinase and Mitogen Activated Protein Kinase families). Within the kinome, GSK3 is most closely related to the Cyclin Directed Kinase (CDK) and Mitogen Activated Protein Kinase (MAPK) groups, with these latter groups sharing a substrate context preference for serine and threonine residues immediately followed (C-terminally) by a proline residue. In contrast, GSK3 shows a different substrate context. It will phosphorylate either serine or threonine residues, with a modestly higher activity against serine being reported (Stamos et al., 2014, Sutherland, 2011). Most importantly GSK3 has strong kinase activity only against serine and threonine residues which are followed 4 amino acids more C-terminally by another phosphorylated serine or threonine residue, in a S/T-X-X-X-pS/pT context (Fig. 1). This activity is described as requiring ‘priming’ by phosphorylation at the C-terminal site. It also allows the sequential phosphorylation by GSK3 of multiple sites each 4 amino acids apart and is a key determinant of GSK3 function. Critically, it means that proteins are not GSK3 substrates until they have themselves been phosphorylated and in many cases the result of this is that the key factors influencing whether GSK3 will phosphorylate a target protein are not factors acting upon GSK3 but rather independent factors acting upon this substrate priming.

The biochemistry of GSK3 and its role in specific areas of physiology and pathology have been reviewed very well elsewhere (Beurel et al., 2015, Forde and Dale, 2007, Hur and Zhou, 2010, Kaidanovich-Beilin and Woodgett, 2011, McCubrey et al., 2014, Ricciardi et al., 2017, Ruvolo, 2017, Sutherland, 2011). Here we will focus on the regulation of GSK3 substrate phosphorylation and its connections with the PI3K-AKT signalling network. In some publications, we feel this latter connection has been over-emphasised and we will discuss the evidence that specific phosphorylation events catalysed by GSK3 are regulated upstream by PI3K and AKT and the interactions of GSK3 with components of the PI3K-AKT-mTOR signalling network.

Section snippets

The regulation of GSK3 activity: what controls whether GSK3 phosphorylates target proteins?

There are several factors which influence the measurable activity of the GSK3 kinase when it is assayed, most notably phosphorylation of the GSK3 N-terminus which will be discussed below. However, the existing data indicate that the dominant factors which control the phosphorylation by GSK3 of its best studied substrates are co-localisation of the kinase with these substrates ensured by protein-binding scaffolds, and the independently regulated priming of these substrates by other protein

GSK3 action within the PI3K-AKT-mTOR signalling network: feedback and crosstalk

A key component of the cell regulatory mechanisms controlling cell growth and proliferation is represented by the PI3K (Class I Phosphoinositide 3-Kinase)-AKT-mTOR (Mechanistic Target Of Rapamycin) signalling network. Many diverse signals including a range of mitogens and growth factors activate cell surface receptors which drive cell growth and proliferation in part through the activation of PI3K, the synthesis of its primary lipid product, PIP3, (phosphatidylinositol 3,4,5-trisphosphate) and

Possible therapeutic use of GSK3 inhibitors: bipolar disorder, Alzheimer's disease, cancer, parasitic infections and more

Lithium salts have been widely used as a mood stabilizer in patients suffering from bipolar disorder for more than 50 years. Although their mechanism of action is still not completely understood, lithium compounds have been found to inhibit GSK-3 in vitro and in vivo (Stambolic et al., 1996; Caberlotto et al., 2013). Lithium, however, also inhibits several other protein kinases and there are many GSK3 independent mechanisms proposed to explain the clinical effects of lithium such as serotonin

Conflict of interest

The authors declare they have no conflict of interest with publication of this manuscript.

Acknowledgements

Work in the NRL laboratory is funded by Medical Research Scotland (1034-2016), Prostate Cancer UK (PG14-006), The Chief Scientist Office (ETM-433), The Brain Tumour Charity (GN-000344) and The PTEN Research Foundation. The authors thank Helen Wise for critical review of the manuscript. MAH is supported by a James Watt Scholarship.

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