Abstract
To investigate the role of PTEN (phosphatase and tensin homolog) in mammalian target of rapamycin complex 2 (mTORC2) signaling in glioblastoma multiforme (GBM), we found higher activation of mTORC2 in PTENmu cells, as evidenced by enhanced phosphorylation of mTOR (Ser2481), AKT (Ser473) and glycogen synthase kinase 3 beta (GSK3β) (Ser9) as compared with PTENwt cells. In addition, PTENwt cells upon PTEN depletion showed mTORC2 activation. The reduced mTORC2 signaling in PTENwt cells was related to higher Rictor phosphorylation at Thr1135 residue. Phosphorylation of Rictor at Thr1135 inhibited its association with mTORC and thus there was a reduction in mTORC2 complex formation. In addition, PTENwt cells expressing mutated Rictor in which Thr1135 was substituted with alanine, showed enhanced mTORC2 formation and signaling. This enhanced mTORC2 signaling promoted inactivation of GSK3β. Thus, we established the reciprocal activation of mTORC2 and GSK3β in GBM. To the best of our knowledge, this is the first report describing role of PTEN in mTORC2 formation by promoting Rictor phosphorylation (Thr1135) in GBM. Furthermore, the drug sensitivity of mTORC2 was evaluated. A newly identified carbazole alkaloid, mahanine, showed cytotoxicity in both PTENmu and PTENwt cells. It inhibited both mTORC1/2 and AKT completely in PTENmu cells, whereas it inhibited only mTORC1 in PTENwt cells. Cytotoxity and AKT-inhibitory activity of the mTORC1/2 inhibitor was increased either by depleting PTEN or in combination with phosphatidylinositol 3 kinase inhibitors in PTENwt cells. In contrast, depletion of Rictor decreased the cytotoxicity of the mTORC1/2 inhibitor in PTENmu cells. Thus, PTEN has an important role in mTORC2 formation and also influences the effectiveness of an mTORC1/2 inhibitor in GBM.
Highlights
MTORC1 contains two more specific subunits namely regulatory-associated protein of mammalian target of rapamycin (Raptor) and PRAS40.6–10 Rapamycininsensitive companion of Mammalian target of rapamycin (mTOR) (Rictor), mSin[1] and protor1/2, are exclusive partners of mammalian target of rapamycin complex 2 (mTORC2).11–13 The binding of Rictor or Raptor to mTOR is mutually exclusive in a certain cellular scenario. mTOR complex 1 (mTORC1) promotes protein translation through activation of
Active mTORC2-specific phosphorylation of mTOR at Ser2481 was higher in PTENmu cells activation of the phosphatidylinositol 3 kinase (PI3K) pathway, which in turn activated mTORC1 as evidenced by increased mTOR phosphorylation at Ser2448 (Figure 1c)
As mTORC1 activity is regulated by the PI3K pathway, we studied the phosphorylation of S6K1
Summary
Mammalian target of rapamycin (mTOR), a serine/threonine (Ser/Thr) kinase protein, has a central role in cell growth and proliferation.[1,2] mTOR complex 1 (mTORC1) and complex 2(mTORC2) are two functionally distinct complexes having some common subunits.[3,4,5] In addition, mTORC1 contains two more specific subunits namely regulatory-associated protein of mammalian target of rapamycin (Raptor) and PRAS40.6–10 Rapamycininsensitive companion of mTOR (Rictor), mSin[1] and protor1/2, are exclusive partners of mTORC2.11–13 The binding of Rictor or Raptor to mTOR is mutually exclusive in a certain cellular scenario. mTORC1 promotes protein translation through activation ofS6K1, inhibition of 4E-BP1 and enhancement of RNA translation via S6 ribosomal protein.[2,14] mTORC2 can phosphorylate AKT at the Ser[473] and take part in cell proliferation, regulation and cytoskeletal reorganization.[15,16] Rapamycin and its analogs (rapalogs) are used for treatment of cancers as mTORC1 inhibitors. MTOR was predominantly associated with Rictor only in PTENmu cells, demonstrating differential mTORC2 formation between phosphorylation (Thr389), that is, the signature of active S6K1 (Figures 2h and i). MTORC1 activity, we knocked down Raptor in the PTENwt cells and observed reduced phosphorylation of both S6K1 (Thr389) and PTEN inhibits mTORC2 formation by stimulating Rictor (Thr1135)
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