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Disclosures
Author contributions
Grants
This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute MERIT Award R37 HL040696 to J.A.F.
Acknowledgments
Introduction
The AGC kinases, named after the protein A, G and C kinases, are an evolutionarily conserved group of proteins that share a Ponesimod australia motif at the c-terminus of their catalytic core. This motif, composed of F-X-X-F/Y-S/T-Y/F, is known as the PIF-pocket and regulates catalytic activity (Arencibia et al., 2013, Manning and Cantley, 2007). The AGC kinase family comprises 14 family members, of which AKT (also known as PKB; protein kinase B) is a key member.
There are three AKT isoforms, transcribed from separate genes, which share three highly conserved domains: a central catalytic domain and two regulatory domains: a lipid-binding N-terminal PH (plektrin homology) domain, and the hydrophobic motif (Fig. 1). The PH domain contains a lipid-binding module that promotes the interaction of AKT with the plasma membrane, an important step in AKT activation. The hydrophobic motif contains an important docking site for the activating kinase PDK1 (3-phosphoinositide-dependent kinase-1) and also provides allosteric regulation of catalytic activity (Scheid & Woodgett, 2003; Fig. 1). There is approximately 80% sequence homology between the isoforms with most variability occurring in the linker region between the PH and catalytic domains (Brodbeck et al., 1999, Cheng et al., 1992, Hanada et al., 2004, Jones et al., 1991).
AKT activation
Mechanisms of AKT activation have been reviewed previously (Liao and Hung, 2010, Scheid and Woodgett, 2003), but essentially, AKT activity is regulated downstream of receptor tyrosine kinases (RTKs), such as those within the EGF (epidermal growth factor), insulin, PDGF (platelet derived growth factor), FGF (fibroblast growth factor) and VEGF (vascular endothelial growth factor) families. RTKs activate class I phosphatidylinositol 3-kinases (PI3K), either directly, or in conjunction with adaptor proteins such as IRS-1/2 (insulin receptor substrate-1/2; Fig. 2). The PI3Ks phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3). AKT binding to PIP3 at the plasma membrane induces a conformational change that results in phosphorylation of AKT, predominantly on two highly conserved residues, Thr308 and Ser473 leading to AKT activation (Fig. 1). Phosphorylation of Thr308 in the activation T-loop of the catalytic domain by PDK1, results in a conformational change that enhances substrate affinity and promotes AKT kinase activity (Alessi et al., 1997). Phosphorylation of Ser473 within the PIF pocket of AKT by mTORC2 (mammalian target of rapamycin complex 2) is thought to promote AKT activity by increasing the affinity of AKT to PDK1 (Sarbassov, Guertin, Ali, & Sabatini, 2005). In fact, multiple different kinases for Ser473 have been described in the literature and it's likely that mechanisms determining full activation of AKT are context dependent. It has been accepted, for example, that following DNA damage, the PI3K-like kinase (PIKK) DNA-PK (DNA-dependent protein kinase) is responsible for AKT Ser473 phosphorylation and that AKT activation prevents apoptosis following ionizing radiation (Bozulic, Surucu, Hynx, & Hemmings, 2008). Multiple other phosphorylation sites on AKT have been described, although the physiological importance of these is not yet fully understood (Risso, Blaustein, Pozzi, Mammi, & Srebrow, 2015) and mechanisms of constitutative activation of AKT signalling in cancer are discussed further below.
Important negative regulators of the PI3K/AKT signalling pathway include the tumour suppressor genes and phosphatases PTEN (phosphatase and tensin homolog), PP2A (protein phosphatase 2A) and PHLPP (PH domain and leucine rich repeat protein phosphatase 1; Gao, Furnari, & Newton, 2005), which dephosphorylate PIP3, AKT pThr308 and AKT pSer473 respectively (Toker & Marmiroli, 2014; Fig. 2). PTEN hydrolyses the 3′-phosphate on PIP3 to terminate PI3K signalling. The SH2 domain-containing inositol phosphatases (SHIP-1/2) are able to hydrolyse the 5′-phosphate on PIP3 to generate PI(3,4)P2, the function of which is not clear, although some studies suggest that like PIP3, PI(3,4)P2 is able to facilitate PDK1 activation of AKT (Gewinner et al., 2009). Recently, a fourth putative tumour suppressor of the PI3K/AKT pathway has been described namely, polyphosphate 4-phosphatase type II (INPP4B; Gewinner et al., 2009). This is able to de-phosphorylate PI(3,4)P2 to PI(3)P in vitro, resulting in attenuation of AKT signalling (Gewinner et al., 2009).