Department of Morphology, Embryology, Human Anatomy Section, Ferrara University, 44100 Ferrara, Italy
Activation. The serine-threonine protein kinase AKT1 is a catalytically inactive cytoplasmic protein. AKT activation occurs by means of stimulation of the growth factor receptor-associated phosphatidylinositol 3-kinase (PI3K) and is a multi-step process that involves both membrane translocation and phosphorylation. When PI3K is activated by either growth factors, cytokines or hormones, PI3K generates 3-phosphorylated phosphoinositides, i.e. phosphatidylinositol-3,4,5-trisphosphate (PIP3) and phosphatidylinositol-3,4-bisphosphate (PIP2) at the plasma membrane. Both phospholipids bind with high affinity to the PH domain, mediating membrane translocation of AKT. At the membrane, AKT1 is phosphorylated at threonine 308 by PDK1 (Andjelkovic et al., 1997; Walker et al., 1998) and at serine 473 by a second kinase identified with mTOR when bound to Rictor in the so called TORC2 complex (Santos et al., 2001; Sarbassov et al., 2005); however, it is still controversial if this second phosphorylation may occur by DNA-dependent protein kinase (Feng et al., 2004; Hill et al., 2002). Other kinases that have been reported to phosphorylate serine 473 are PKC (Kawakami et al., 2004), integrin-linked kinase (ILK) (Troussard et al., 2003; Lynch et al., 1999; Delcommenne et al., 1998), MAP kinase-activated protein kinase-2 (MK2) (Rane et al., 2001), PDK-1 (Balendran et al., 1999) or Akt itself (Toker et al., 2000). The full activation of AKT1 requires phosphorylation at both sites; threonine 308 phosphorylation increases the enzymatic activity up to 100-fold and serine 473 phosphorylation by a further 10-fold, thus both phosphorylation events enhance AKT1 activity by 1000-fold (Kumar et al., 2005; Alessi et al., 1996). The activation is rapid and specific, and it is abrogated by mutations in the AKT PH domain. Once activated, AKT1 dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus.
Beside these essential activation sites, threonine 72 and serine 246 residues undergo auto-phosphorylation (Li et al., 2006), serine 124 and threonine 450 residues are constitutively phosphorylated, while tyrosine 315 and 326 in the activation loop can be phosphorylated by Src kinase, maybe regulating AKT1 activity (Chen et al., 2001).
Regulation. AKT activation is inversely regulated by phosphatases: PH domain leucine-rich repeat protein phosphatase (PHLPP) dephosphorylates the serine 473 residue of AKT1 (Brognard et al., 2007), and protein phosphatase 2 (PP2) dephosphorylates the threonine 308 residue (Gao et al., 2005). PI(3,4,5)P3 is hydrolyzed by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Src homology domain-containing inositol phosphatases SHIP1/SHIP2. PTEN antagonizes PI3K activity by removing the phosphate at the D3 position generating PI(4,5)P2 (Maehama et al., 1998), while SHIP1/2 dephosphorylates the D5 position to produce PI(3,4)P2 (Deleris et al., 2003; Damen et al., 1996).
Apoptosis inhibition. Survival factors can suppress apoptosis and enhance survival of cells by activating AKT, which inactivates components of the apoptotic machinery. AKT directly regulates apoptosis by phosphorylating and inactivating pro-apoptotic proteins such as bad, which controls release of cytochrome c from mitochondria, caspase-9, which after AKT dependent phosphorylation promotes cell survival (Donepudi et al., 2002; Downward et al., 1999; Franke et al., 2003) and apoptosis signal-regulating kinase-1 (ASK1), a mitogen-activated protein kinase involved in stress- and cytokine-induced cell death that, once phosphorylated on serine 83, reduces apoptosis (Autret et al., 2008; Datta et al., 1997; Del Peso et al., 1997; Zha et al., 1996). The pro-survival proline-rich AKT substrate of 40kDa (PRAS40) can be phosphorylated on threonine 246, attenuating its ability to inhibit mTORC1 kinase activity (Van der Haar, 2007). PRAS40 appears to protect neuronal cells from apoptosis after stroke (Kovacina et al., 2003) and has been proposed to promote cell survival in cancer cells (Huang et al., 2005).
Proliferation. AKT can stimulate cell cycle progression through the inhibitory phosphorylation of the cyclin-dependent kinase inhibitors p21 and p27 (Viglietto et al., 2002; Liang et al., 2002; Shin et al., 2002; Zhou et al., 2001; Rossig et al., 2001). The AKT dependent inhibition of GSK3 stimulates cell cycle progression by stabilizing cyclin D1 expression (Diehl et al., 1998). AKT activation can promote progression through mitosis, even in the presence of DNA damage (Kandel et al., 2002); a mechanism explaining this observation is that AKT directly phosphorylates the DNA damage checkpoint kinase Chk1 on serine 280 (King et al., 2004), blocking checkpoint function by stimulating Chk1 translocation to the cytosol. With no K protein kinase-1 (WNK1) seems to be a negative regulatory element in the insulin signaling pathway that regulates cell proliferation. AKT phosphorylates WNK1 on threonine 60 within the AKT consensus sequence (Vitari et al., 2004). The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, with growth-suppressive function. AKT phosphorylates merlin on threonine 230 and serine 315 residues, abolishing binding partners and leading to merlin degradation by ubiquitination (Tang et al., 2007).
Metabolism. AKT phosphorylates the GSK3alpha and GSK3beta isoforms, which are involved in metabolism regulation by decreasing glycogen synthesis and increasing glycolytic enzymes transcription (Jope et al., 2004; Kohn et al., 1996), thus relating AKT activation with high glycolysis efficiency in cancer cells (Warburg effect). AKT1 is also involved in tolerance of cells to nutrient depletion, allowing tumor progression under hypovascular conditions (Izuishi et al., 2000). The TBC1 domain family member 1 (TBC1D1), AKT substrate phosphorylated on threonine 590, may be involved in controlling GLUT1 glucose transporter expression through the mTOR/p70S6K pathway (Zhou et al., 2008). The Rab-GAP AS160 (also known as TBC1D4) has emerged as an important direct target of AKT involved in GLUT4 translocation to the plasma membrane (Sano et al., 2003). In hepatocytes, AKT can also inhibit gluconeogenesis and fatty acid oxidation through direct phosphorylation on serine 570 of PGC-1alpha (Li et al., 2007), which is a gene coactivator with FoxO1 and other transcription factors.
Angiogenesis. AKT plays important roles in angiogenesis through effects in both endothelial cells and cells producing angiogenic signals. AKT activates endothelial nitric oxide synthase (eNOS) through direct phosphorylation on the serine 1179 site, resulting in increased production of nitric oxide (NO) in vascular endothelium, which stimulates vasodilatation, vascular remodelling and angiogenesis (Iantorno et al., 2007).
Translation. A well known AKT substrate is the serine/threonine kinase mammalian target of rapamycin (mTOR), which controls the translation of several proteins important for cell cycle progression and growth (Starkman et al., 2005; Varma et al., 2007). AKT can directly phosphorylate and activate mTOR, as well as cause indirect activation of mTOR by phosphorylating two sites on the tuberous sclerosis complex 2 (TSC2) tumour suppressor protein, also called tuberin (Manning et al., 2002). mTOR forms two complexes : TORC1 , in which mTOR is bound to Raptor, and TORC2, in which mTOR is bound to Rictor. In the TORC1 complex, mTOR signals to its downstream effectors S6 kinase/ribosomal protein and 4EBP-1/eIF-4E to control protein translation. In the TORC2 complex, mTOR can phosphorylate AKT itself thus providing a positive feedback on the pathway (Sarbassov et al., 2005). The mTOR effector S6 kinase-1 (S6K1) can also regulate the pathway by inhibiting the insulin receptor substrate (IRS), thus preventing IRS proteins from activating the PI3K/AKT signaling (Harrington et al., 2004; Shah et al., 2004). The Y box-binding protein 1 (YB-1) is a DNA/RNA-binding protein through the Y-box motif in target sequences. AKT phosphorylates YB-1 on serine 102, leading to an enhancement of cap-dependent translation of multidrug resistance 1 (MDR1) gene (Bader et al., 2008).
Nuclear functions. Among the AKT substrates identified into cell nucleus, acinus is a nuclear factor required for chromatin condensation which induces resistance to caspases proteolysis and to apoptosis when phosphorylated by AKT on serine 422 and 573 (Hu et al., 2005). Phosphorylation of the murine double minute 2 (MDM2/HDM2 in humans) oncogene by AKT promotes its translocation to the nucleus, where it negatively regulates p53 function with subsequent modification of the cell cycle in relation to DNA repair mechanisms (Vousden et al., 2002; Mayo et al., 2005). Several Akt substrates are nuclear transcription factors: AKT blocks forkhead transcription factors (FKHR/FOXO1) and in particular the FoxO subfamily-mediated transcription of genes that promote apoptosis, cell cycle arrest and metabolic processes. When phosphorylated by AKT, FKHR are sequestrated in the cytoplasm thus inhibiting transcription (Nicholson et al., 2002; Datta et al., 1997). AKT can phosphorylate IKK, indirectly increasing the activity of nuclear factor kappa B (NF-kB), which stimulates the transcription of pro-survival genes and regulates the immunity response (Ozes et al., 1999; Romashkova et al., 1999; Verdu et al., 1999). The cAMP-response element binding protein (CREB) is a direct target for phosphorylation by AKT, occurring on a site that increases binding of CREB to proteins necessary for induction of genes containing cAMP responsive elements (CREs) in their promoter regions; CREB has been shown to mediate AKT-induced expression of antiapoptotic genes bcl-2 and mcl-1 (Du et al., 1998). AKT can regulate the telomerase activity necessary for DNA replication; recombinant AKT was found to enhance telomerase activity by phosphorylating the human telomerase reverse transcriptase (hTERT) subunit, which contains a consensus motif as AKT substrate. The helix-loop-helix transcription factor tal1, required for blood cell development, is specifically phosphorylated by AKT at threonine 90, causing its nuclear redistribution (Palamarchuk et al., 2005b). Insulin induces GATA2 phosphorylation on serine 401 by AKT. GATA2 transcription factor is an inhibitor of adipogenesis and activator of vascular cells. AHNAK is a protein of exceptionally large size localized into nuclei and able to shuttle between nucleus and cytoplasm; it is downregulated in several tumors (Amagai et al., 2004). It has been reported that in epithelial cells its extranuclear localization is regulated by AKT dependent phosphorylation (Sussman et al., 2001). ALY is a nuclear speckle protein implicated in mRNA export. The PI3K/AKT signaling regulates its subnuclear residency, cell proliferation, and mRNA export activities through nuclear AKT dependent phosphorylation on threonine 219 and phosphoinositide association (Okada et al., 2008). AKT specifically phosphorylates serine 350 of the Nur77 protein within its DNA-binding domain, decreasing its transcriptional activity by 50-85% and connecting the AKT axis with a nuclear receptor pathway (Pekarsky et al., 2001). The breast cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein that acts as a tumor suppressor; heregulin induces AKT-dependent phosphorylation of BRCA1, which has been implicated in altering its function (Altiok et al., 1999).
Orthologs. AKT is evolutionarily conserved in eukaryotes ranging from Caenorhabditis elegans to man. The amino acid identity between C. elegans and human AKT1 is around 60%; the mouse AKT1 is 90% homologous to human AKT1 at the nucleic acid level and 98% homologous at the amino acid level (Hanada et al., 2004; Bellacosa et al., 1993).
For details see : HomoloGene.
Also the phosphorylation sites on the AKT substrates are conserved amongst the orthologs from all mammals; this evolutionary conservation can be indicative of the relevance of the substrate toward the AKT cellular functions.
Daniela Etro ; Silvia Missiroli ; Francesca Buontempo ; Luca Maria Neri ; Silvano Capitani
AKT1 (v-akt murine thymoma viral oncogene homolog 1)
Atlas Genet Cytogenet Oncol Haematol. 2009-05-01
Online version: http://atlasgeneticsoncology.org/gene/355/akt1