As currently understood, the mTORC1-dependent feedback mechanism affecting PI3K-Akt signaling acts upstream of PI3K on the IRS proteins. However, attenuation of growth-factor stimulated Akt phosphorylation is seen more generally upon disruption of the TSC1-TSC2 complex, such as under full serum growth conditions [27
], where the IRS proteins should play only a minor role. In studies of MEFs lacking Tsc1
, we observe loss of Akt phosphorylation on Thr-308, Ser-473, and, to a lesser extent, the turn motif at Thr-450 (JH and BDM, unpublished), suggesting that multiple inputs into Akt activation are impaired.
Given the complexity of Akt activation and the strong mTORC1-driven feedback inhibition upstream of PI3K in these cells, we decided to directly assay mTORC2 kinase activity following its isolation via immunoprecipitation of Rictor [30
]. Using an exogenous Akt substrate to measure mTORC2 kinase activity, we found that mTORC2 activity was severely blunted in Tsc1
- or Tsc2
-deficient MEFs or HeLa cells with stable shRNA-mediated knockdown of TSC2
. Reciprocally, overexpression of TSC2 in wild-type cells increased mTORC2 kinase activity in these assays. Importantly, insulin-stimulated mTORC2 activity was not restored to Tsc2
-deficient MEFs after prolonged rapamycin treatment or siRNA-mediated knockdown of Rheb or Raptor, despite all of these treatments blocking mTORC1 activity and restoring insulin signaling to PI3K. Furthermore, phosphorylation of Akt on Ser-473 was also reduced in Tsc2
null cells, relative to reconstituted cells, following expression of a variety of constitutively active PI3K alleles, which should bypass feedback mechanisms acting upstream. Finally, stable expression of a GAP-dead mutant of TSC2 partially restored mTORC2 activity and Akt phosphorylation to Tsc2−/−
MEFs, without detectable effects on the constitutive mTORC1 signaling in these cells. Collectively, these data demonstrate that the TSC1-TSC2 complex promotes mTORC2 activity in a manner that is independent of Rheb, mTORC1, and mTORC1-mediated feedback effects on PI3K.
In further support of the TSC1-TSC2 complex being involved in mTORC2 activation, we have recently found reduced levels of hydrophobic motif phosphorylation on PKCα (Ser-657) and total PKCα levels in Tsc2−/−
MEFs (JH and BDM, unpublished). While not as severe, these effects on PKCα are similar to that described for MEFs lacking the mTORC2 components Rictor, Sin1, or mLST8 [10
]. Unlike Akt, PKCα does not appear to be affected by feedback loops stemming from mTORC1 activation, and in fact, we found that prolonged rapamycin treatment further decreased PKCα phosphorylation in the Tsc2−/−
cells (JH and BDM, unpublished). These results provide further support that mTORC2 activity is defective in Tsc2
−/− cells through mechanisms other than mTORC1-dependent feedback or cross-talk with mTORC2.