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. 2017 Dec 14;171(7):1545-1558.e18.
doi: 10.1016/j.cell.2017.10.037. Epub 2017 Nov 16.

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling

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Free PMC article

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling

Gina Lee et al. Cell. .
Free PMC article

Abstract

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Keywords: CK1; RNA splicing; RNA stability; S6K1; SR proteins; SRPK2; cancer metabolism; de novo lipid synthesis; mTOR; nonsense-mediated decay.

Figures

Figure 1
Figure 1. Identification of SRPK2 as a downstream target of mTORC1
(A) The SILAC-based phospho-proteomics analysis was performed on Tsc2−/− MEFs treated with vehicle or rapamycin (20 nM) for 2 hr. MS (Top) and MS/MS (Bottom) spectra of TVS*ASS*TGDLPK peptide from SRPK2 (asterisks indicate sites of phosphorylation) are shown. (B) Schematics of SRPK2 protein domains (Top) and amino acid conservation (Bottom). The mTORC1-dependent phosphorylation sites are highlighted in red. (C) Immunoblot analysis of HEK293E cells treated with insulin (100 nM) with or without rapamycin (Rapa, 20 nM) pre-treatment for 30 min after overnight serum starvation. (D) Immunoblot analysis of HEK293E cells transfected with siRNAs targeting SRPK2 or control. Cells were serum starved overnight and treated with insulin (100 nM) for 2 hr with or without rapamycin (20 nM) pre-treatment for 30 min. (E, F) Immunoblot analysis of cancer cell lines treated with rapamycin (100 nM) for 24 hr with serum starvation. pSRPK2(S494) detects two bands in these cells.
Figure 2
Figure 2. SRPK2 phosphorylation at Ser494 by S6K1 primes Ser497 phosphorylation by CK1
(A) Alignment of SRPK2 amino acid sequence with AGC kinase phosphorylation motif. (B) Immunoblot analysis of HEK293E cells transfected with siRNAs targeting S6K1 or control. Cells were treated with insulin (100 nM) for 2 hr after overnight serum starvation. (C) Immunoblot analysis of HEK293E cells. Cells were serum starved overnight and treated with the indicated small molecule inhibitors for 30 min, followed by insulin (100 nM) treatment for 2 hr. Rapamycin (100 nM), Torin1 (250 nM), MK2206 (10 µM), and PF4708671 (10 µM) were used. (D) Immunoblot analysis of HEK293E cells transfected with HA-S6K1-CA (constitutively active S6K1) or vector. Cells were serum starved overnight and treated with insulin (100 nM) for 2 hr with or without rapamycin (20 nM) pre-treatment for 30 min. (E) HEK293E cells transfected with HA-S6K1 were serum starved overnight and treated with insulin (100 nM) for 2 hr with or without rapamycin (20 nM) pretreatment for 30 min. In vitro kinase essay was performed using HA-S6K1 immunoprecipitated (IP) from these cells. Recombinant GST-SRPK2-wild-type (WT) and S494A proteins were used as substrates. WCL, whole cell lysates. (F, G) Immunoblot analysis of LAM 621-101 cells expressing HA-SRPK2-WT, S494A, or S497A. Endogenous SRPK2 was knocked down with shSRPK2 targeting 3’ UTR. (H) Alignment of SRPK2 amino acid sequence with Casein sequences corresponding to the conserved CK1 substrate motif. (I) In vitro CK1 kinase assay with recombinant GST-SRPK2-S494D, S494A, and S494A/S497A proteins as substrates.
Figure 3
Figure 3. mTORC1-dependent phosphorylation of SRPK2 induces its nuclear translocation and SR protein phosphorylation
(A) Immunostaining of SRPK2 (green) in HEK293E cells transfected with siRNAs targeting SRPK2 or control. Cells were serum starved overnight and treated with insulin (100 nM) for 2 hr with or without Torin1 (250 nM) pre-treatment for 30 min. Right panels show the enlarged images of the white boxes in the left panels. DAPI (blue), nucleus. Scale bar, 50 µm. (B) Immunoblot analysis of HEK293E cells treated with insulin (100 nM) for 2 hr after overnight serum starvation. Half of the cell lysates were treated with CIAP to dephosphorylate proteins. (C) Immunostaining of SRPK2 (green) and pS6-S235/S236 (red) in the indicated cell lines treated with rapamycin (20 nM) or Torin1 (250 nM) for 2 hr. DAPI (blue), nucleus. Scale bars, 10 µm. (D) Top, Immunostaining of SRPK2 (green) in LAM 621-101 cells (TSC2−/−) reconstituted with empty vector or TSC2. Right panels show the enlarged images of the white boxes in the left panels. DAPI (blue), nucleus. Scale bar, 50 µm. Bottom, Immunoblot analysis of the reconstituted cells. (E) Immunoblot analysis of cytoplasmic (Cyto) and nuclear fractions (Nuc) of LAM 621-101 cells treated with rapamycin (100 nM) for 4 hr after overnight serum starvation. (F) Immunostaining of SRPK2 (white) in LAM 621-101 cells expressing SRPK2-WT or S494A. Endogenous SRPK2 was knocked down with shSRPK2 targeting 3’ UTR. Cells were treated with vehicle or rapamycin (20 nM) for 2 hr. Right panels show the enlarged images of the white boxes in the left panels. Scale bar, 50 µm. (G) Quantification of nuclear-cytoplasmic distribution of SRPK2 in (F). The number of cells counted is indicated. (H) Immunoblot analysis of LAM 621-101 cells transfected with HA-SRPK2-WT, K110M (kinase dead), or S494A. (I) Quantification of the average band intensity of phosphorylated SR proteins normalized to GAPDH in (H). n = 3. *p < 0.05. See also Figure S1.
Figure 4
Figure 4. mTORC1 and SRPK2 signaling regulates expression of genes involved in de novo lipid synthesis
(A) Venn diagrams of the differentially regulated transcripts identified from the whole transcriptome microarray analysis on LAM 621-101 cells. One analysis was conducted on the conditions where cells were treated with rapamycin (20 nM) or vehicle for 24 hr. The second analysis was conducted on the conditions where cells stably express shRNAs targeting SRPK2 or GFP. Fold cut-off for the gene expression change (linear fold change) or splicing index (SI) is ≥ 1.5 or ≥ 2, respectively. SI = [Condition 1 (Probe intensity/Gene intensity)] / [Condition 2 (Probe intensity/Gene intensity]. n = 3. P < 0.05. (B) Fold decreases of 21 commonly downregulated genes identified from the microarray analysis. Genes involved in lipid metabolism are highlighted in blue. (C) Schematics of de novo lipogenesis. Genes identified from the microarray are highlighted in red. (D) qPCR analysis of lipogenic genes in LAM 621-101 cells. Cells were treated with vehicle, rapamycin (20 nM), or Torin1 (250 nM) for 24 hr with serum starvation (left). Cells stably expressing shRNAs targeting SRPK2 or GFP were serum starved overnight (right). n = 3. *p < 0.05. (E) Immunoblot analysis of LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP. Cells were treated with rapamycin (20 nM) or vehicle for 24 hr with serum starvation. (F) Immunoblot analysis of LAM 621-101 cells treated with rapamycin (100 nM) or vehicle for 24 hr with serum starvation. SRPK2 KO, SRPK2 CRISPR knockout cells. (G) Immunoblot analysis of LAM 621-101 cells stably expressing mouse Srpk2-WT (wild type) or AA (S488A/S491A which corresponds to S494A/S497A in human SRPK2). Cells were transfected with siRNAs targeting SRPK2 or control and serum-starved overnight. See also Figure S2.
Figure 5
Figure 5. mTORC1 and SRPK2 signaling regulates mRNA stability of lipid biosynthetic genes
(A) Promoter activity analysis of LAM 621-101 cells transfected with promoter constructs with siRNAs targeting SREBP1 and SREBP2 (siSREBP1/2) or control in the presence or absence of serum. Promoter activity measured by renilla luciferase was normalized by cypridine luciferase. n = 2. (B) Promoter activity analysis of LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP in the absence of serum. n = 2. (C) Immunoblot analysis of nuclear fractions or whole cell lysates (WCL) in LAM 621-101 cells in the presence or absence of serum. FL-SREBP1, full-length SREBP1. (D) Measurement of mRNA stability in LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP. Cells were serum starved for 24 hr with rapamycin (100 nM) or vehicle treatment, followed by actinomycin D (Act D, 5 µg/ml) treatment for the indicated time points. qPCR was performed to measure mRNA levels of the indicated genes. n = 3. (E) qPCR analysis of WT or SRPK2 knockout (KO) LAM 621-101 cells transfected with siRNAs targeting UPF1 or control. n = 3. (F) qPCR analysis of LAM 621-101 cells transfected with siRNAs targeting each SRSF or control. n = 3. (G) Immunoblot analysis of HEK293E cells transfected with empty vector or SRSF1-V5. Cells were treated with Torin1 (250 nM) or vehicle for 4 hr. Immunoprecipitation (IP) was performed with anti-V5 antibody. 2% total cell lysate was loaded as an input. (H) qPCR analysis of LAM 621-101 cells transfected with siRNAs targeting U1-70k or control. n = 3. *p < 0.05 and **p < 0.01. NS, not significant. See also Figures S3 and S4 and Tables S1 and S2.
Figure 6
Figure 6. SRPK2 is necessary for de novo fatty acid and cholesterol synthesis
(A) Left, Schematic of radioactive 14C-acetate incorporation assay for de novo fatty acid and cholesterol synthesis. Cells were cultured in serum-free medium. Right, HPLC analysis of radio-labeled fatty acids and cholesterol in LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP. (B) Quantification of integrated peak areas in (A) normalized to internal standard 13(S)-HODE. n = 2. (C, E) Immunoblot analysis of LAM 621-101 cells transfected with siRNAs targeting SRPK2 or control (C). Cells were treated with rapamycin (20 nM) or Torin1 (250 nM) for 24 hr (E). (D, F) Radioactive 14C-acetate incorporation into fatty acids and cholesterol in LAM 621-101 cells transfected with siRNAs targeting SRPK2 or control (D). Cells were treated with rapamycin (20 nM) or Torin1 (250 nM) for 24 hr (F). Graphs represent quantification of integrated HPLC peak areas normalized to internal standard 13(S)-HODE. n = 2. (G) LC-MS analysis of de novo fatty acid synthesis from U-13C-glucose. LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP were serum starved for 48 hr. Left, Schematics of de novo fatty acid synthesis. Right, Graph represents de novo synthesized fatty acids (FA). n = 4. Arbitrary unit (AU). (H) Immunoblot analysis of LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP. Cells were grown without or with 10% dialyzed FBS (dFBS) or lipoprotein-deficient serum (LPDS) for 48 hr. (I, J) LC-MS analysis of de novo fatty acid synthesis from U-13C-glucose. LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP were grown with 10% dFBS (I) or LPDS (J). Graphs represent de novo synthesized fatty acids. n = 4. *p < 0.05. See also Figure S5.
Figure 7
Figure 7. Inhibition of SRPK2 blunts cell growth upon lipid starvation
(A, C) Crystal violet staining of LAM 621-101 cells stably expressing shRNAs targeting SRPK2 or GFP. Lipoprotein (25 µg/ml), oleate-albumin (50 µM; oleate), or fatty acid-free albumin (25 µM; control) was supplemented to the media. (B, D) Quantification of (A) and (C), respectively. (E) Immunoblot analysis of WT or SRPK2 knockout (KO) LAM 621-101 cells treated with SRPIN340 (30 µM) for 48 hr with serum starvation. (F) Immunoblot analysis of LAM 621-101 cells transfected with siRNAs targeting SRPK1, SRPK2, or control. (G) Crystal violet staining of the indicated cell lines treated with SRPIN340 at the indicated concentrations. (H) Xenograft tumor growth assays of WT (control) or SRPK2 knockout (KO) LAM 621-101 cells. Graph represents the fold change of tumor size relative to week 0 (week 0 = tumor formation). n = 8 tumors. (I–K) Xenograft tumor growth assays of RT4 (I) and ELT3 (Tsc2−/− rat leiomyoma)-luciferase cells (J, K) treated with SRPIN340. (I) Graph represents the fold change of tumor size relative to week 0. (J) Bioluminescent imaging of mice bearing ELT3-luciferase tumors. (K) Graph represents the fold change of total photon flux relative to week 0 in (J). n = 6–8 tumors. *p < 0.05. See also Figures S6 and S7.

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