GPCR signaling inhibits mTORC1 via PKA phosphorylation of Raptor
- University of Texas Southwestern Medical Center, United States
- University of California, San Diego, United States
- Moores Cancer Center, University of California San Diego, United States
- Children's Hospital and Institutes of Biomedical Sciences, Fudan University, China
- University of California San Diego, United States
Figures
Figure 1 with 2 supplements
Increased levels of cAMP inhibit mTORC1 activation and global protein synthesis.
(A) Overexpression screen identifying GPCRs involved in mTORC1 regulation. GPCRs were overexpressed in human embryonic kidney 293A (HEK293A) cells, and 24 h later mTORC1 activity was analyzed by …
Figure 1—figure supplement 1
Protein levels of the mTORC1 complex or small G-proteins involved in mTORC1 signaling are unaffected by forskolin.
(A) Schematic presentation of mTORC1 including mTOR, Raptor, mLST8, Deptor, and PRAS40 and upstream regulators. The small G-protein Rheb activates mTORC1 downstream of growth factor signaling, …
Figure 1—figure supplement 2
mTORC1 inhibition via increased levels of cAMP is not upstream of the tuberous sclerosis complex.
(A) Growth factors, energy status, stress, and amino acids converge on mTORC1 to regulate its activity. (B) Forskolin inhibits mTORC1 and not mTORC2. HEK293A cells were treated with 10 μM forskolin …
Figure 2
Protein Kinase A mediates the effect of cAMP to inhibit mTORC1.
(A) Inhibition of PKA by H89 blocks the effect of forskolin on mTORC1. Human embryonic kidney 293A (HEK293A) cells were pretreated with or without the protein kinase A (PKA) inhibitor H89, and then …
Figure 3
cAMP suppresses amino acid-induced mTORC1 activation.
(A) Forskolin inhibits mTORC1 activation by insulin. Human embryonic kidney 293A (HEK293A) cells were starved of fetal bovine serum (FBS) for 16 h. After FBS starvation cells were starved of amino …
Figure 4
Amino acid-induced mTORC1 lysosomal localization is not blocked by cAMP.
(A) Forskolin does not block amino acid-induced mTOR lysosomal localization. Immunofluorescence analysis depicting mTOR (green) and LAMP2 (red) in mouse embryonic fibroblasts (MEFs). Merged depicts …
Figure 5
cAMP induces Raptor phosphorylation.
(A) Forskolin increases Raptor phosphorylation detected by a phospho-PKA substrate antibody. Empty vector, Flag-tagged mTOR, Flag-tagged Raptor, Myc-tagged PRAS40, or Myc-tagged mLST8 was expressed …
Figure 6 with 5 supplements
Protein kinase A phosphorylates Raptor at Ser 791.
(A) Forskolin stimulates Ser 791 (S791) phosphorylation in Raptor. HA empty vector, HA-tagged Raptor, HA-tagged Raptor S791A, HA-tagged Raptor S791D, HA-tagged Raptor S792A, or HA-tagged Raptor …
Figure 6—figure supplement 1
Forskolin inhibits mTORC1 in the absence of AMPK.
(A) Forskolin inhibits mTORC1 in AMPK knockout MEF cells. AMPK α1/2 wild-type (AMPK +/+) or AMPK α1/2 knockout (KO) (AMPK -/-) MEFs were treated with or without forskolin and mTORC1 activity was …
Figure 6—figure supplement 2
Raptor Ser 791 phosphorylation does not alter Raptor binding to the Rag GTPases.
HEK293A cells were treated with or without 10 μM forskolin. The following plasmids were overexpressed (+) or not overexpressed (-). HA-tagged Raptor, HA-tagged Raptor S791A, HA-tagged Raptor S791D, …
Figure 6—figure supplement 3
cAMP does not alter mTORC1 kinase activity or binding of mTORC1 components.
(A) cAMP doesn't alter mTORC1 kinase activity. HA-tagged Raptor or HA-tagged Raptor mutants (S791A, S791D, S792D, S791D/S792D) were overexpressed with Myc-tagged mTOR in HEK293A cells. Forty-eight ho…
Figure 6—figure supplement 4
Generation of the Raptor S791A mutant HEK293A cells using CRISPR/Cas9 genome editing.
(A) Generation of Raptor S791A cells. Sequence depicting the locus in the RPTOR gene showing the single guide RNA (sgRNA, green), the 5’NGG protospacer adjacent motif (PAM; blue), and the mutation …
Figure 6—figure supplement 5
Raptor Ser 791 phosphorylation decreases mTORC1 activity and cell proliferation.
(A) Raptor Ser 791 phosphorylation decreases mTORC1 activity. Right: HEK293A or HEK293A Raptor S791A mutant cells (S791A-1 or S791A-2) were treated with or without forskolin and mTORC1 activity was …
Figure 7
Activation of Gαs-coupled GPCRs inhibit mTORC1 activity in vivo.
(A) MDA-MB-231 cells were pretreated with or without the protein kinase A (PKA) inhibitor H89, and then treated with or without 10 μM forskolin, 10 μM epinephrine, 10 μM isoprenaline, or 10 μM …
Author response image 1
Author response image 2
Sequencing chromatogram for knock-in clone S791A-1.
Author response image 3
Sequencing chromatogram for knock-in clone S791A-2.
Author response image 4
Sequencing chromatogram for heterozygous knock-in.
3 peaks can be observed in the chromatogram.For example, for the nucleotides underlined in red, you can see 3 distinct peaks for the first 2 nucleotides.
Author response image 5
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.43038.016
