Phosphorylation of Rictor at Thr1135 impairs the Rictor/Cullin-1 complex to ubiquitinate SGK1

Abstract

The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism, cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase. However, its upstream regulatory pathways as well as whether it has additional function(s) remain largely unknown. We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1, but not with other Cullin family members. Furthermore, we identified SGK1 as its downstream target. Interestingly, Rictor, but not Raptor or mTOR, promotes SGK1 ubiquitination. As a result, SGK1 expression is elevated in Rictor^--/-- MEFs. We further defined that as a feedback mechanism, Rictor can be phosphorylated by multiple AGC family kinases including Akt, S6K and SGK1. Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1. On the other hand, it disrupted the interaction between Rictor and Cullin-1. Consequently, T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction. This finding further expands our knowledge of Rictor’s function. Furthermore, our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades, and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.

Keywords

mTORC2 / Rictor / kinase / E3 ubiqutin ligase / Cullin-1 / SGK1

References

[1] Carrano, A.C., Eytan, E., Hershko, A., and Pagano, M. (1999). SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1 , 193–199 .10.1038/12013
[2] Gao, D., Wan, L., Inuzuka, H., Berg, A.H., Tseng, A., Zhai, B., Shaik, S., Bennett, E., Tron, A.E., Gasser, J.A., . (2010). Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction. Mol Cell 39, 797–808 .10.1016/j.molcel.2010.08.016
[3] 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 .10.1042/BJ20081668
[4] Ghosh, P., Wu, M., Zhang, H., and Sun, H. (2008). mTORC1 signaling requires proteasomal function and the involvement of CUL4-DDB1 ubiquitin E3 ligase. Cell Cycle 7, 373–381 .10.4161/cc.7.3.5267
[5] Guertin, D.A., and Sabatini, D.M. (2007). Defining the role of mTOR in cancer. Cancer Cell 12, 9–22 .10.1016/j.ccr.2007.05.008
[6] Gwinn, D.M., Shackelford, D.B., Egan, D.F., Mihaylova, M.M., Mery, A., Vasquez, D.S., Turk, B.E., and Shaw, R.J. (2008). AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30, 214–226 .10.1016/j.molcel.2008.03.003
[7] Huang, J., Wu, S., Wu, C.L., and Manning, B.D. (2009). Signaling events downstream of mammalian target of rapamycin complex 2 are attenuated in cells and tumors deficient for the tuberous sclerosis complex tumor suppressors. Cancer Res 69, 6107–6114 .10.1158/0008-5472.CAN-09-0975
[8] Jacinto, E., Facchinetti, V., Liu, D., Soto, N., Wei, S., Jung, S.Y., Huang, Q., Qin, J., and Su, B. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127, 125–137 .
[9] Loffing, J., Flores, S.Y., and Staub, O. (2006). Sgk kinases and their role in epithelial transport. Annu Rev Physiol 68, 461–490 .10.1146/annurev.physiol.68.040104.131654
[10] Lu, Z., Xu, S., Joazeiro, C., Cobb, M.H., and Hunter, T. (2002). The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. Mol Cell 9, 945–956 .10.1016/S1097-2765(02)00519-1
[11] Maddika, S., and Chen, J. (2009). Protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase. Nat Cell Biol 11, 409–419 .10.1038/ncb1848
[12] Majumder, P.K., and Sellers, W.R. (2005). Akt-regulated pathways in prostate cancer. Oncogene 24, 7465–7474 .10.1038/sj.onc.1209096
[13] Manning, B.D., and Cantley, L.C. (2007). AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274 .
[14] Park, J., Leong, M.L., Buse, P., Maiyar, A.C., Firestone, G.L., and Hemmings, B.A. (1999). Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18, 3024–3033 .10.1093/emboj/18.11.3024
[15] Reiling, J.H., and Sabatini, D.M. (2006). Stress and mTORture signaling. Oncogene 25, 6373–6383 .10.1038/sj.onc.1209889
[16] Sabatini, D.M. (2006). mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 6, 729–734 .10.1038/nrc1974
[17] Sahoo, S., Brickley, D.R., Kocherginsky, M., and Conzen, S.D. (2005). Coordinate expression of the PI3-kinase downstream effectors serum and glucocorticoid-induced kinase (SGK-1) and Akt-1 in human breast cancer. Eur J Cancer 41, 2754–2759 .10.1016/j.ejca.2005.07.018
[18] Samuels, Y., Wang, Z., Bardelli, A., Silliman, N., Ptak, J., Szabo, S., Yan, H., Gazdar, A., Powell, S.M., Riggins, G.J., . (2004). High frequency of mutations of the PIK3CA gene in human cancers. Science 304, 554.10.1126/science.1096502
[19] Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L., and Sabatini, D.M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 .10.1126/science.1157535
[20] Sarbassov, D.D., Ali, S.M., Sengupta, S., Sheen, J.H., Hsu, P.P., Bagley, A.F., Markhard, A.L., and Sabatini, D.M. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22, 159–168 .10.1016/j.molcel.2006.03.029
[21] Sarbassov, D.D., Guertin, D.A., Ali, S.M., and Sabatini, D.M. (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 .10.1126/science.1106148
[22] Shiota, C., Woo, J.T., Lindner, J., Shelton, K.D., and Magnuson, M.A. (2006). Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev Cell 11, 583–589 .10.1016/j.devcel.2006.08.013
[23] Wullschleger, S., Loewith, R., and Hall, M.N. (2006). TOR signaling in growth and metabolism. Cell 124, 471–484 .
[24] Yang, Q., and Guan, K.L. (2007). Expanding mTOR signaling. Cell Res 17, 666–681 .10.1038/cr.2007.64