Differential requirement of CAAX-mediated posttranslational processing for Rheb localization and signaling

Abstract

The Rheb1 and Rheb2 small GTPases and their effector mTOR are aberrantly activated in human cancer and are attractive targets for anti-cancer drug discovery. Rheb is targeted to endomembranes via its C-terminal CAAX (C=cysteine, A=aliphatic, X=terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl isoprenoid. After farnesylation, Rheb undergoes two additional CAAX-signaled processing steps, Ras converting enzyme 1 (Rce1)-catalyzed cleavage of the AAX residues and isoprenylcysteine carboxyl methyltransferase (Icmt)-mediated carboxylmethylation of the farnesylated cysteine. However, whether these postprenylation processing steps are required for Rheb signaling through mTOR is not known. We found that Rheb1 and Rheb2 localize primarily to the endoplasmic reticulum and Golgi apparatus. We determined that Icmt and Rce1 processing is required for Rheb localization, but is dispensable for Rheb-induced activation of the mTOR substrate p70 S6 kinase (S6K). Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks Rheb localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of Rheb. We conclude that inhibitors of Icmt and Rce1 will not block Rheb function, but FTS could be a promising treatment for Rheb- and mTOR-dependent cancers.

Figure 1. Rheb1 and Rheb2 are localized primarily to the ER and Golgi. COS-7 cells were transiently transfected with pEGFP vector or with pEGFP encoding GFP-tagged Rheb1 or Rheb2

(a) Rheb localization in cis- and trans-Golgi and associated endomembranes. Live cells were imaged after staining with BODIPY TR C₅ ceramide. (b) Rheb localization in cis-Golgi. Cells were fixed and immunostained with mouse anti-GM130 antibody followed by Alexa Fluor 594-conjugated anti-mouse secondary antibody. (c) Rheb localization in the ER. Cells were fixed and immunostained with rabbit anti-ERp72 antibody followed by Alexa Fluor 594-conjugated anti-rabbit secondary antibody. All images were acquired on a Zeiss confocal microscope using LSM software. Data shown are representative of more than 10 images from at least two independent experiments.

Figure 2. CAAX-signaled modifications alone are sufficient for Rheb1 but not Rheb2 subcellular localization to the ER and Golgi

(a) Comparison of C-terminal membrane targeting sequence elements of H-Ras, K-Ras4B, Rnd3, Rheb1, and Rheb2. Palmitoylated cysteines (in H-Ras) and polybasic residues (in K-Ras 4B and Rnd3) are underlined. The C-terminal CAAX motifs are shaded. (b) GFP fusion proteins terminating in the Rheb1 or Rheb2 C-terminal sequences shown were encoded by cDNA sequences subcloned into the pEGFP vector. (c) NIH 3T3 cells were transiently transfected with pEGFP vector, or with pEGFP encoding the following proteins: GFP-tagged full-length Rheb1 C181S (Rheb1 SAAX), full-length Rheb2 C180S (Rheb2 SAAX), the indicated C-terminal Rheb1 or Rheb2 sequences, or full-length (FL) Rheb1 or Rheb2. Live cells were imaged by confocal microscopy. Data shown are representative of two independent experiments.

Figure 3. Rheb subcellular localization is dependent on Rce1- and Icmt-catalyzed modifications

(a) Rce1 +/+, Rce1 −/−, Icmt +/+, and Icmt −/− MEFs were transiently transfected with pEGFP vector or with pEGFP encoding GFP-tagged Rheb1 (F-Rheb1), Rheb1 M184L (GG-Rheb1), Rheb2 (F-Rheb2), or Rheb2 M183L (GG-Rheb2). Live cells were imaged by confocal microscopy. (b) Quantification of the data shown in (a). At least 30 cells in each condition were scored.

Figure 4. Rce1- and Icmt-catalyzed modifications are not required for Rheb1 activation of S6K

(a) Wild-type (Icmt +/+) Rce1 −/−, and Icmt −/− mouse embryo fibroblasts were transiently transfected with pEGFP vector or with pEGFP encoding Rheb1 Q64L (F-Rheb1 64L), Rheb1 M184L/Q64L (GG-Rheb1 64L), or Rheb1 C181S/Q64L (Rheb1-SAAX 64L), along with pRK7 HA-S6K1, and serum- and amino-acid starved as described in Materials & Methods. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K, HA, GFP, or β-actin (loading control). (b) Wild-type (Icmt +/+), Rce1 −/−, and Icmt −/− MEFs were transiently transfected with pcDNA3 vector, or with pcDNA3 encoding HA-tagged K-Ras G12V, FLAG-tagged Rheb1 N153T, or AU1-tagged mTOR E2419K, along with pRK7 HA-tagged S6K1, and serum- and amino acid-starved as in (a). Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K, HA, Rheb1, K-Ras, or β-actin (loading control). (c) Rce1 +/+, Rce1 −/−, Icmt +/+, and Icmt −/− MEFs were stably infected with either non-specific shRNA (NS) or shRNA targeting mouse TSC2 (TSC2 sh). Cells were lysed 5 d post-infection and lysates were immunoblotted with antibodies to TSC2, phospho-S6K, total S6K, phospho-S6, total S6, or Rheb1. Data shown are representative of at least two independent experiments.

Figure 5. FTS does not disrupt Rheb or H-Ras subcellular localization

(a) NIH 3T3 cells were transiently transfected with pEGFP vector, pEGFP encoding GFP-tagged Rheb1, or pEYFP encoding YFP-tagged H-Ras and treated with DMSO (Vehicle), 5 µM FTI-2153, or 75 µM FTS overnight. Live cells were imaged by confocal microscopy.

Figure 6. FTS inhibits mTOR-induced S6K activation by a mechanism independent of prenylated proteins

(a) NIH 3T3 cells were transiently transfected with pcDNA3 vector, or with pcDNA3 encoding HA-tagged K-Ras G12V, FLAG-tagged Rheb1 N153T, or AU1-tagged mTOR E2419K, along with pRK7 HA-tagged S6K1. Three h following transfection, medium was replaced with complete medium containing either DMSO (Vehicle) or 5 µM FTI-2153. Twenty-four h following transfection, cells were serum-starved in DMEM supplemented with 0.1% BSA containing DMSO, 5 µM FTI-2153, or 75 µM FTS overnight, and then amino-acid starved in PBS containing the indicated compound for 1 h. Cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies to phospho-S6K, HA, Rheb1, K-Ras, or β-actin (loading control). Data are representative of three independent experiments. (b) NIH 3T3 cells were transiently transfected with the indicated plasmids, as described above. Three h following transfection, medium was replaced with complete medium supplemented with DMSO, 10 µM FTI-2153, 20 µM GGTI-2417, or 10 µM FTI + 20 µM GGTI. Cells were serum- and amino-acid starved as in (a). Cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies to phospho-S6K, HA, Rheb1, K-Ras, Rap1, or β-actin (loading control). (c) NIH 3T3 cells were transiently transfected with the indicated plasmids. Three h following transfection, medium was replaced with complete medium supplemented with DMSO (Vehicle), 10 µM FTI-2153, 20 µM lovastatin, or 30 µM lovastatin. Serum- and amino acid-starved cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies to phospho-S6K, HA, Rheb1, K-Ras, H-Ras, or β-actin (loading control). Data shown are representative of at least two independent experiments.

Figure 7. FTS blocks endogenous S6K activation in MCF-7 cells

MCF-7 cells growing in complete medium were treated with 100 µM FTS in complete medium for 0, 2, 6, 16, or 24 h. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K, total S6K, phospho-S6, total S6, mTOR, and β-actin (loading control). Data shown are representative of two independent experiments.