UNA PROPUESTA PARA EL PATRIMONIO CULTURAL.

also known as PKB (protein kinase B) and RACPK (related to A and C protein kinases) (Bellacosa et al.,1990; Jones et al., 1991). AKT kinases belong to the AGC kinase family, related to AMP/GMP kinases and protein kinase C. The three conserved domains they consist of are an N-terminal pleckstrin homology (PH) domain, a central kinase catalytic (CAT) domain, and a C-terminal extension (EXT) containing a regulatory hydrophobic motif (HM). The PH domains are ~80% identical among AKT isomers and ~30% identical to PH domains in pleckstrin and other proteins. The PH domain is connected to the CAT domain by the linker (LINK) region which is poorly conserved among the AKT isoforms (17– 46% identical) and has no significant homology to any other human protein (Kumar and Madison, 2005). The consensus CAT domains are ~90% identical among the AKT isoforms and are closely related the PKC, protein kinase A (PKA), serum-and glucocorticoid-induced protein kinase (SGK), and S6 sub families of the AGC kinase family whilst the C-terminal EXT is ~70% identical among the AKT isoforms and is most closely related to the PKC family (Kumar and Madison, 2005). AKT is regulated by both phosphorylation and the direct binding of PI3K lipid products to its PH domain. However, PI3K independent mechanisms to activate AKT have been identified as well (Burgering and coffer,1995; Franke et

al., 1995).

Phosphorylation of phosphatidylinositol-4,5-biphosphate (PI3,4,5-P2) by PI3K generates phosphatidylinositol-3,4,5-triphosphate (PI3,4,5-P3) which in turns binds with PH domain of AKT protein and phosphatidylinositol-dependent protein kinase (PDK1) recruiting them to the membrane. Akt activation involves the phosphorylation of two residues, Thr308 and Ser 473. Thr308 is phosphorylated by PDK1 in the activation loop and Ser473 is phosphorylated by PDK2 localising into c-terminal HM (Franke, 2008). mTOR forms part of two different complexes which are mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)

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needed for regulating different proteins. Upstream mTORC2 is essential to phosphorylate AKT whereas mTORC1 acts downstream of AKT (Zoncu et al., 2011). AKT inhibits its substrates including the members of the Forkhead family of transcription factors and glycogen synthase kinase-3 (GSK-3). Inhibition of GSK-3 induces the up-regulation of the anti-apoptotic protein MCL-1 (Maurer et

al., 2006). The phosphorylation of the Forkhead family member FoxO3a and

GSK-3 is induced and MCL-1 protein is increased by the activation of PI3K/AKT pathway in CLL. Inhibition of PI3K induces loss of cell viability, dephosphorylation of FoxO3a and GSK-3, and decrease in the level of MCL-1 protein (Ringshausen

et al., 2002; Petlickovski et al., 2005). Higher capacity for cell cycle progression

is associated with a stronger activation of AKT pathway in CLL cells from patients with progressive disease (Longo et al., 2007). This group has demonstrated the importance of activation of AKT in cell cycle progression and proliferation of CLL cells. When specific pharmacological inhibitors are used for PI3K/AKT/mTOR, ERK and JNK pathways, the complete inhibition of the proliferative response was observed indicating the importance of these signalling pathways in cell survival of CLL. Nevertheless, constitutively activated AKT kinases can increase leukaemic cell viability and expression of the anti-apoptotic proteins Mcl-1, Bcl- xL and X-linked inhibitor of apoptosis protein (XIAP). Longo et al., (2007) also demonstrated that the strength of AKT, ERK and/or JNK activation determines the different proliferative responses in various CLL subsets. Survival of CLL cells is also promoted by AKT upon activation by several pro-survival signals such as IL-4, phorbol ester, human albumin and immobilised anti-IgM (Barragan et al., 2002; Jones et al., 2003). AKT phosphorylation was inhibited by PI3K inhibitor, LY294002 and PKC inhibitor suggesting the existence of two different pathways for AKT phosphorylation in CLL cells (Barragan et al., 2006).

AKT is expressed on macrophages regulate their survival pathway. AKT-1 is constitutively activated in human macrophages and the addition of PI3K inhibitor, LY294002 suppressed the activation of AKT-1 and induced cell death confirming constitutive activation of AKT was regulated by PI3K serine/threonine kinase. It was also reported that inhibition of PI3K or use of a dominant negative (DN) AKT- 1 to suppress AKT-1 resulted in loss of mitochondrial membrane potential and thereby activation of caspase-9 and -3 and DNA fragmentation in mice (Hongtao

et al.,2001). PI3K/AKT signalling pathway is also present in the lipid rafts of

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AKT activation. The synthetic antitumour lipids (ATLs), are a family of compounds which can induce apoptosis in tumour cells and they could inhibit PI3K/AKT survival signals by dephosphorylating AKT and displacing AKT and key enzyme regulators from lipid rafts (Reis-Sobreiro et al., 2013). AKT inhibitors are used to inhibit the phosphorylation of AKT thereby to inhibit cell survival and cell cycle progression. A-443654 is a potent, ATP-competitive and reversible inhibitor of AKT catalysed phosphorylation activity. It is a pan-AKT inhibitor which effect on AKT1, AKT2 or AKT3 (Luo et al., 2005; Shi et al., 2005). It was reported that A- 443654 inhibits other protein kinases as well including PKA, protein kinase C- related kinase 2 (PRK2), mitogen- and stress-activated protein kinase1 (MSK1) and dual-specificity tyrosine-phosphorylated and -regulated kinase1A (DYRK1A) showing less selectivity. An ATP non-competitive AKT inhibitor, Akti-1/2 is a highly selective AKT inhibitor, which blocks AKT1 and AKT2 but not AKT3 activity. Akti- 1/2 requires the PH domain for its activity (Barnett et al., 2005; Logie et al., 2007). Novel specific inhibitor AiX can antagonise AKT activation by direct blockade and induces apoptosis in U-CLL cells (Hofbauer et al., 2010).