RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells

Abstract

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.

Keywords: 4E-BP1; 70kDa ribosomal protein S6 kinase 1; Akt; PRAS40; RAPTOR; RAPTOR-independent companion of mTOR; RICTOR; Sin1; TSC; TSC2; ULK1; UTR; caRags; caRheb; caRhoA; constitutively active Rags B and C; constitutively active Rheb; constitutively active RhoA; eukaryotic initiation factor 4E binding protein 1; mTOR; mTORC; mTORC2; mechanistic target of rapamycin; mechanistic target of rapamycin complex; p70S6K1; proline-rich Akt/PKB substrate 40kDa; regulatory-associated protein of mTOR, complex 1; stress-activated map kinase-interacting protein 1; tuberous sclerosis complex; uncoordinated-51-like kinase 1; untranslated region; wild type RhoA; wtRhoA.