Биологические науки/5. Молекулярная биология

¹Bulgakova O.V., ¹Bersimbay R.I., ²Sarbassov D.

¹Department of Natural Sciences, The L.N. Gumilyov Eurasian National University, Astana, 010008, Kazakhstan.

²Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

The PI3K/Akt/mTOR signaling pathway

Deregulation of growth factor signaling pathway is common in human cancers. The recent sequencing studies of multiple human tumor samples strengthen this relationship by indicating that the genes encoding the components of growth factor signaling are mutated in high frequency [1,2]. Following many years of studying growth factor signaling pathways, it has been defined that this signaling pathway plays a crucial role in regulation of cell proliferation, survival, and differentiation.

One of the crucial downstream effector of PI3K identified as the Akt kinase, also known as PKB (protein kinase B) and it is activated by its translocation to the plasma membrane and phosphorylation [3].

Akt belongs to a large AGC (protein kinase A, G, and C) kinase family and within total of 518 human protein kinases this family is represented by 60 members of the highly conserved and essential kinases. The AGC kinases are defined and classified by their sequence homology to the kinase domains of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), and protein kinase C (PKC). Among the members of this kinase family, Akt is the evolutionarily conserved serine/threonine kinase and an essential downstream effector of the PI3K pathway in growth factor signaling that act on a wide spectrum of substrates [4]. Two Akt genes are found in nematode Caenorhabditis elegans, whereas in mammals Akt is represented by subfamily of kinases containing three isoforms expressed by the distinct genes, Akt1, Akt2, and Akt3. It indicates that in evolution the rising complexity of growth factor signaling impelled duplication of the Akt gene. All Akt isoforms are highly related structurally and represented by three well-defined domains.

The full length of human Akt1 encoded by the polypeptide of 480 amino acids. It contains the N-termianal pleckstrin homology (PH) domain located within the amino acids 6 to 107 that plays a critical role in the functional translocation of Akt to the plasma membrane and its activation. The catalytic kinase domain resides within the amino acids 154 to 477 and its functional activity is regulated by the activation segment located within the kinase domain positioned from residue 219 to 314 also known as activation loop. Phosphorylation of the activated loop on the Thr-308 site required for the Akt kinase activity. The turn motif phosphorylation site resides on the Thr-450 site. Another stretch of amino acids within residue 469 to 474 is identified as the hydrophobic motif (HM) found in a non-catalytic region following kinase domain also involved in regulation of the kinase activity linked to phosphorylation of the hydrophobic Ser-473 site.

Within the AGC kinase members Akt represents one of the highly regulated members of this family. The detailed functional and structural studies of Akt led to a basic understanding how its known domains and motifs determine the PI3K-dependent regulation of the kinase activity of Akt.

The phosphorylation-dependent regulation is common among the multiple AGC kinase members. In regulation of Akt the phosphorylation of the three sites are critical in switching on the Akt kinase to active mode. The phosphorylation of Akt on Thr-308 and Ser-473 sites is dependent on the growth factor/PI3K signaling, whereas its Thr-450 site is constitutively phosphorylated. Following growth factor stimulation and translocation of Akt to the plasma membrane location, Akt is phosphorylated on the Thr-308 and Ser-473 sites, the key phosphorylation sites, required to fully activate Akt. The activation loop of Akt on Thr-308 is phosphorylated by the phosphoinositide-dependent kinase 1 (PDK1) that required for the kinase activity of Akt [5]. PDK1 as the Akt kinase was identified in 1997, whereas the regulatory Ser-473 kinase of Akt, named PDK2, was identified following several years only in 2005 as the mTOR Complex 2 (mTORC2) [6]. Although several candidates were proposed earlier as PDK2 [7], the mouse genetic studies confirmed the role of mTORC2 as the Ser-473 kinase of Akt [8]. Interestingly, the constitutively phosphorylated site of Akt on Thr-450 known as the turn motif site is also dependent on mTORC2, but in this case it is not dependent on growth factor signaling. The mechanism of this regulation remains poorly characterized. Recently, the DNA protein kinase (DNA PK) activated following the DNA damage response has been also shown to phosphorylate AKT on its hydrophobic Ser-473 site [9].

The mTORC2 substrate Akt positively regulates mTORC1, whereas the mTORC1 substrate S6K1 has a negative effect on the PI3K/mTORC2/Akt signaling by inducing rapid turnover of IRS1, which causes attenuation of the insulin/IGF signaling [10].

Akt as the AGC kinase family carries the well-defined hydrophobic motif on its C-terminuis that initiated to study a role of mTORC2 in regulation of Akt. In the present time based on the distinct location of the hydrophobic motif site within the AGC kinase family members, mTORC2 has been defined as a major regulatory kinase of Akt [6] and also other essential member of this family of kinases known as SGK [11]. Regulation of Akt by its phosphorylation on the hydrophobic Ser-473 site is coupled to activation of the growth factor dependent PI3K signaling. The functional role of mTORC2 as the Ser-473 kinase of Akt defines this kinase complex as the component of growth factor signaling.

How growth factor signaling regulates the mTORC2 kinase activity remains very poorly understood. The distinct growth factor dependent phosphorylation of the mTORC2 components mTOR and rictor has been identified. The phosphorylation of mTOR on its hydrophobic Ser-2481 site is linked to the active state of mTORC2 [12]. This site of mTOR has been previously characterized and defined as its hydrophobic autophosphorylation site. Why this autophosphorylation site reflects a high kinase activity specifically of mTORC2, but not mTORC1, is not known. The essential component of mTORC2 rictor is a phosphoprotein. Several distinct phosphorylation sites have been identified on rictor and its Thr-1135 site has been characterized as the phosphorylation site regulated by growth factor signaling. Interestingly, S6K1 is a kinase of the Thr-1135 site on rictor implying its potential role as a coordination link between the two distinct mTOR complexes. This site has been shown to carry a negative role in regulation of mTORC2 by providing the binding site for the 14-3-3 adaptor proteins [13]. The functional studies indicated that the rictor Thr-1135 site is not essential in regulation of the mTORC2 kinase activity and this phosphorylation takes place independent of mTORC2 [14]. Until now, how growth factor signaling regulates the mTORC2 kinase activity is unknown. Understanding of the mTORC1 regulation linked to activity of the G-protein rheb is much more advanced compare to our understanding how the second mTOR complex is regulated. It is possible that similar to mTORC1, the regulation of mTORC2 is also G-protein dependent and identification of this low affinity binding regulatory protein remains elusive in the present time.

The Akt signaling is engaged in multiple signaling pathways and involved in regulation of a variety of cellular processes, including cell proliferation, growth, survival, and metabolism. Perturbations of the Akt signaling are associated with human diseases including cancer and the metabolic syndrome. Several Akt substrates are functionally relevant to human cancer.

REFERENCES

1. Ding, L., Getz, G., Wheeler, D.A., Mardis, E.R., McLellan, M.D., Cibulskis, K., Sougnez, C., Greulich, H., Muzny, D.M., Morgan, M.B., et al. (2008). Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455, 1069-1075.

2. Parsons, D.W., Jones, S., Zhang, X., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., et al. (2008). An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807-1812.

3. Shaw, R.J., and Cantley, L.C. (2006). Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441, 424-430.

4. Bellacosa, A., Kumar, C.C., Di Cristofano, A., and Testa, J.R. (2005). Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv Cancer Res 94, 29-86.

5. Alessi, D.R., James, S.R., Downes, C.P., Holmes, A.B., Gaffney, P.R., Reese, C.B., and Cohen, P. (1997). Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7, 261-269.

6. Sarbassov, D.D., Guertin, D.A., Ali, S.M., and Sabatini, D.M. (2005b). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098-1101.

7. Dillon, R.L., White, D.E., and Muller, W.J. (2007). The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer. Oncogene 26, 1338-1345.

8. Guertin, D.A., Stevens, D.M., Thoreen, C.C., Burds, A.A., Kalaany, N.Y., Moffat, J., Brown, M., Fitzgerald, K.J., and Sabatini, D.M. (2006). Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell 11, 859-871.

9. Bozulic, L., Surucu, B., Hynx, D., and Hemmings, B.A. (2008). PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell 30, 203-213.

10. Guertin, D.A., and Sabatini, D.M. (2005). An expanding role for mTOR in cancer. Trends Mol Med 11, 353-361.

11. Garcia-Martinez, J.M., and Alessi, D.R. (2008). mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J 416, 375-385.

12. Copp, J., Manning, G., and Hunter, T. (2009). TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res 69, 1821-1827.

13. Dibble, C.C., Asara, J.M., and Manning, B.D. (2009). Characterization of Rictor phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1. Mol Cell Biol.

14. Boulbes, D., Chen, C.H., Shaikenov, T., Agarwal, N.K., Peterson, T.R., Addona, T.A., Keshishian, H., Carr, S.A., Magnuson, M.A., Sabatini, D.M., et al. Rictor Phosphorylation on the Thr-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2. Mol Cancer Res.