www.sciencesignaling.org/cgi/content/full/9/430/ra57/dc1 Supplementary Materials for The 4E-BP eif4e axis promotes rapamycinsensitive growth and proliferation in lymphocytes Lomon So, Jongdae Lee, Miguel Palafox, Sharmila Mallya, Chaz G. Woxland, Meztli Arguello, Morgan L. Truitt, Nahum Sonenberg, Davide Ruggero, David A. Fruman* The PDF file includes: *Corresponding author. Email: dfruman@uci.edu Published 31 May 2016, Sci. Signal. 9, ra57 (2016) DOI: 10.1126/scisignal.aad8463 Fig. S1. Acute inhibition of S6K activity is dispensable for lymphocyte growth and proliferation. Fig. S2. Hypomorphic genetic model shows that S6K activity is dispensable for lymphocyte growth and proliferation. Fig. S3. Specific inactivation of mtor kinase activity in lymphocytes phenocopies complete mtor or mtorc1 loss in lymphocytes. Fig. S4. Amino acid sequence surrounding the Thr 37 phosphoacceptor site on 4E- BP1 and 4E-BP2 is distinct and conserved. Fig. S5. Gene expression patterns of eif4ebp1 and eif4ebp2 are distinct specifically in mature lymphocytes. Fig. S6. Rapamycin partially inhibits eif4f complex formation in lymphoma cells and MEFs. Fig. S7. Rapamycin has a lesser effect on the proliferation of MEFs than that of MLN0128. Fig. S8. M can be inducibly expressed in naïve lymphocytes and is sufficient to block growth and proliferation equivalent to rapamycin or TOR-KIs. Fig. S9. M blocks lymphocyte growth and proliferation in vivo. Fig. S10. M inhibits only proliferation without affecting size in lymphoma cells. Table S1. Accession numbers for species analyzed.
Fig. S1. Acute inhibition of S6K activity is dispensable for lymphocyte growth and proliferation. (A) The percentages of peripheral T cell subsets and B220 + B cells in wild-type (WT) and S6K2-deficient (S6K2 / ) mice were measured by flow cytometry. Left: Representative plots from the analysis of splenocytes. Right: Pooled data from three spleens of each strain. Data are means ± SEM of three mice per genotype. (B) CFSE-labeled CD4 + T cells (top two rows) and B cells (bottom two rows) were stimulated in the absence or presence of 500 nm S6K1i or 20 nm
rapamycin for 24 hours, and S6K activity was measured by flow cytometric analysis of ps6 staining. For each cell type, the top row represents WT cells and the bottom row shows S6K2 / cells. The red numbers indicate the percentages of cells that stained positive for ps6. (C) The growth and proliferation of the cells described in (B) were measured as described in Fig. 1. Results are representative of at least three independent experiments. (D) CFSE-labeled total splenocytes from WT and S6K1/2 / mice were left unstimulated or were stimulated with SEB in the absence or presence of rapamycin for 96 hours. The proliferation of gated CD4 + cells was assessed by flow cytometric analysis of CFSE dilution. The peak corresponding to non-divided cells under stimulatory conditions represents CD4 + T cells with TCRs that did not respond to SEB. Results are representative of at least three independent experiments.
A C S6K1 Kinase-Switch (KS) Analog-sensitive kinase allele (ASKA) mutation G101S/Y102F/L172A Hypomorphic activity: S6K1 activity x S6K1 KS/KS S6K2 -/- S6K1 KS/KS S6K2 -/- ~90% reduction in all S6K activity CD4+ T cell (αcd3/28) WT Stim Rap 20nM KS/KS S6K1 S6K2 73.9 0.9 10.7 -/- B Cell # CD8 % of Max 47.7 48.1 40.1 18.9 WT 23.6 17.2 S6K2 -/- S6K1 KS/KS WT S6K1 KS/KS S6K2 -/- 24.8 23.2 B220 27 CD4 12.7 52 44.9 19.7 WT S6K1 KS/KS S6K2 -/- 14.4 Unstim Stim Rap 24.4 p-s6 25.7 98.9 89.1 Cell size (FSC) CFSE % of Max CFSE D B cell (αigm+il4) WT KS/KS Stim Rap 20nM S6K1 S6K2 p-s6 74.4 2.05 10.2 -/- % of Max WT S6K1 KS/KS S6K2 -/- Cell size (FSC) Unstim Stim Rap CFSE % of Max CFSE Fig. S2. Hypomorphic genetic model shows that S6K activity is dispensable for lymphocyte growth and proliferation. (A) A S6K1 knock-in mouse model harboring the analog-sensitive kinase allele (ASKA: three amino acid substitutions in the kinase domain) of S6K1 (KS: kinase-switch) was crossed to S6K2 deficient mice. (B) Peripheral T cell subsets and B220+ B cells were measured by flow cytometry. (C and D) S6K1 KS lymphocytes show severe hypomorphic activity as intracellular staining for p-s6 shows near complete loss of S6K activity in S6K1 KS S6K2 -/- lymphocytes. Cells growth and proliferation of S6K1 KS S6K2 -/- lymphocytes were examined as in main Figure 1. Results represent at least two independent experiments.
Fig. S3. Specific inactivation of mtor kinase activity in lymphocytes phenocopies complete mtor or mtorc1 loss in lymphocytes. (A) Representative genotyping of the mtor KI mice. (B) Excision PCR analysis of purified CD4 + T cells from mice of the indicated genotypes (Δ: deletion product). The KI allele with the D2338A mutation was amplified by the PCR reaction as a WT (+) product. Dashed red vertical lines were added as a visual aid to distinguish between the indicated genotypes. (C) Lymphocytes from the indicated mice were labeled with CFSE, stimulated as described earlier, and analyzed by flow cytometry to measure ps6 abundance. The experiment shown is separate from the experiment show in Fig. 3B. (D) The percentages of CD4 +
T cells and B220 + B cells in the spleens of mice of the indicated genotypes were measured by flow cytometric analysis. Data are means ± SEM of 4 to 11 mice per genotype. **P < 0.01; ***P < 0.001, by repeated-measures analysis of variance measured versus the medium control. (E) The indicated mice were injected intraperitoneally with PBS or SEB. Forty-eight hours later, cell proliferation was assessed as described in Fig. 3D. Data are means ± SEM of three or four mice per genotype. *P < 0.05, by repeated-measures analysis of variance measured versus the PBS control. (F) efluor670-labeled total splenocytes from mice of the indicated genotypes were activated in vitro with SEB for 96 hours. The proliferation of gated CD4 + T cells was determined by flow cytometric analysis. Results are representative of cells from three or four mice of each genotype.
A Mus musculus Rattus norvegicus Cricetulus griseus Pongo abelii Gorilla gorilla Homo sapiens Macaca mulatta Ophiophagus hannah -5 T36 Mus musculus Rattus norvegicus Cricetulus griseus Pongo abelii Gorilla gorilla Homo sapiens Macaca mulatta Ophiophagus hannah 4E-BP2-5 T37 B WT V R I G31H G31Q V R I V R I C ps6 pakt Akt /2 DKO WT WT G31H G31Q V R I V R I V R I V R I γ β expressed α γ β endogenous α γ β α Actin Suppl Fig4 Fig. S4. Amino acid sequence surrounding the Thr 37 phosphoacceptor site on and 4E-BP2 is distinct and conserved. (A) Sequence alignments of and 4E-BP2 proteins from various species. ClustalW2 software from EMBL-EBI (www.ebi.ac.uk) was used for the analysis. The 5 position (depicted with a light blue arrow) upstream of the Thr 36 or Thr 37 phosphoacceptor sites of or 4E-BP2 (depicted with red arrows) is indicated. The accession number for each species is provided in table S1. (B) mutants (G31H or G31Q) were generated by site-directed mutagenesis and were introduced into either WT or /2 DKO MEFs. Cells were left untreated (V, vehicle) or were treated 20 nm rapamycin (R) or 100 nm MLN0128 (I) for 3 hours before being analyzed by Western blotting with antibodies against the indicated proteins. 4E-BP phosphorylation was assessed by the mobility shift that resulted in the appearance of three different forms (α, β, and γ) upon mtor inhibition. Phosphospecific antibodies were not used for these experiments out of concern that their affinities for the altered sequences might be different. Results are representative of at least five independent experiments.
Percentile of expression Percentile of expression 100 50 0 Eif4ebp1 () HSC preprob Fo B CD4+ CD8+ -50-100 100 80 60 40 20 Eif4ebp2 (4E-BP2) HSC preprob Fo B CD4+ CD8+ 0 Suppl Fig5 Fig. S5. Gene expression patterns of eif4ebp1 and eif4ebp2 are distinct specifically in mature lymphocytes. The expression patterns of eif4ebp1 and eif4ebp2 during hematopoiesis were searched in the Stanford Gene Expression Commons database (https://gexc.stanford.edu).
A m7gtp cap OCI-Ly1 VAL Vehicle Rap 128 Vehicle Rap 128 eif4g eif4e input B m7gtp cap WT MEF DKO MEF V Rap 128 Torin V Rap 128 Torin eif4g eif4e pakt eif4g eif4e 4E-BP2 eif4g input C m7gtp cap WT MEF 0.1% 10% Rap Torin DKO MEF 0.1% 10% Rap Torin pakt 4EBP1 4EBP2 eif4g eif4e input eif4g eif4e pakt Suppl Fig6 Fig. S6. Rapamycin partially inhibits eif4f complex formation in lymphoma cells and MEFs. (A) The indicated DLBCL cell lines were left untreated or were treated for 2 hours with the indicated inhibitors before being subjected to m7gtp cap pulldown assays and Western blotting analysis with antibodies against the indicated proteins. (B) MEFs from WT and /2 DKO mice were left untreated or were treated with the indicated inhibitors before being subjected to m7gtp pulldown assays and Western blotting analysis to show the 4E-BP dependency of the TOR-KIs. (C) To mimic the quiescent state of primary lymphocytes, WT and /2 DKO MEFs were serum-starved in medium containing 0.1% FCS for 24 hours and then were pre-treated with the indicated inhibitors for 15 min before being incubated in medium containing 10% FCS for 2 hours. The cells were then subjected to m7gtp cap assays and Western blotting analysis with antibodies against the indicated proteins. Data in all panels are representative of two independent experiments.
Intensity (% control of MEF) Cell number A 10% FBS 0.1% FBS 10% FBS stimulation 400000 300000 200000 Vehicle Rapamycin MLN0128 * 400000 300000 200000 Vehicle Rapamycin MLN0128 * 100000 ** * 100000 ** * B 0 0 0 24 48 72 (h) 0 24 48 72 (h) Lymphocytes MEF B CD4 T 3T3 Ly1 1x 0.5x 0.25x 0.125x 1.5 eif4e Erk 1.0 0.5 Lymphocytes 0.0 B CD4 T MEF Human CD4 T Mouse CD4 T Ly1 Ly8 VAL MEF 1.5 eif4e 1.0 * Erk 0.5 0.0 Human CD4 T MEF Fig. S7. Rapamycin has a lesser effect than on the proliferation of MEFs than that of MLN0128. (A) Left: Primary MEFs were left untreated (Vehicle) or were treated with the indicated inhibitors, and proliferation was assessed by cell counting at the indicated times. Right: To mimic the quiescent state of lymphocytes, MEFs were treated as described for fig. S6C and then cell numbers were assessed by cell counting at the indicated times. Data in both panels are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01, by repeated-measures analysis of variance measured versus the medium control or by comparing rapamycin- and MLN0128-treated cells. (B) Left: Primary B cells and CD4 + T cells from WT mice, the indicated DLBCL lymphoma cell lines, and the indicated relative amounts of MEFs were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of four independent experiments. Right: The relative amounts of eif4e protein in the indicated cells relative to that in MEFs were determined by densitometric analysis. Data are means SEM of four experiments.
Fig. S8. M can be inducibly expressed in naïve lymphocytes and is sufficient to block growth and proliferation equivalent to rapamycin or TOR-KIs. (A) CD4 + T and B cells from the indicated mice were incubated with doxycycline (Dox, 1 μg/ml) for 6 hours, and M abundance was analyzed by Western blotting. M is FLAG-
tagged and has a higher molecular mass than that of endogenous. (B) B cells from the indicated mice were incubated with the indicated concentrations of doxycyclin for 6 hours before being stimulated for 24 hours and then analyzed by Western blotting with the indicated antibodies to assess mtorc2-akt signaling. Control samples were B cells from Rosa26-rtTA mice treated with or without doxycyclin. (C) Left: M expressing CD4 + T cells (top) and B cells (bottom) were labeled with CFSE and stimulated as indicated for 24 hours. mtorc1-s6k signaling was assessed by intracellular staining and flow cytometric analysis of ps6 (S240/244). Rapamycin served as a control to fully ablate the generation of ps6. Right: Cell proliferation was assessed by flow cytometric analysis of CFSE dilution at 48 hours after stimulation. (D) CD4 + T cells (left) and B cells (right) from the indicated mice were stimulated as described earlier in the absence or presence of the indicated inhibitors. Cell proliferation at 72 hours after stimulation was assessed by flow cytometric analysis of CFSE dilution. Data in all panels are representative of four independent experiments.
CD69 (MFI) FSC Vβ8 A R26-rtTA or M CD4+ T cell (efluor labeled) i.v. C57/B6 SEB i.p. Gate on CD4+Vβ8+eFluor+ CD4 12.5 efluor 3.29 CD4 27.1 B 24h 400 300 200 100 Unstim - + - + 0 R26-rtTA M Dox Fig. S9. M blocks lymphocyte growth and proliferation in vivo. (A) Schematic representation of the adoptive transfer experiment shown in Fig. 8D. (B) Median fluorescence intensity (MFI) of the early T cell activation marker CD69 at 24 hours after injection of the indicated mice with SEB. Data from the experiment shown in Fig. 8D and are means SEM of three mice per group.
Cell proliferation (% of control) % of Max Cell proliferation (% of control) Protein/Cell (% of control) A B NIH/3T3 EV M - + - + 150 100 OCI-Ly1 Dox Actin 1.5 1.0 0.5 0.0 - + Rap - + EV Vehicle Rapamycin M ** *** M Dox 2.0 1.5 1.0 0.5 0.0 * n.s. - + Rap - + EV M Dox 50 0 - + Rap - + EV M Dox Size (FSC-H) Supple Fig9 Fig. S10. M inhibits only proliferation without affecting size in lymphoma cells. (A) Left: NIH/3T3 cells engineered to express M upon treatment with doxycyclin or transfected with empty vector (EV), which served as a negative control, were analyzed by Western blotting with antibodies against the indicated proteins. Middle: Cell proliferation was measured at 72 hours after doxycyclin treatment by cell counting and was normalized to the counts of control samples (untreated cells). Right: Total protein content per cell was measured by a standard Bradford assay and divided by the total cell number at 72 hours. (B) OCI-Ly1 cells were engineered to express M upon treatment with doxycyclin or were transfected with EV as a negative control. Left: Cell proliferation was measured as described in (A). Cell size was measured 24 hours after doxycyclin treatment by flow cytometric analysis of FSC. Results are representative of at least three independent experiments. Where indicated, data are means ± SEM of three or four experiments. *P < 0.05; **P < 0.01; ***P < 0.001, by repeated-measures analysis of variance measured versus EV with dox control.
Table S1. Accession numbers for species analyzed. Species Accession Code 4E-BP2 Accession Code Mus musculus NP_031944.3 CAG47036.1 Rattus norvegicus NP_446309.1 NP_001244839.1 Cricetulus griseus XP_003498263.1 XP_004049592.1 Pongo abelii XP_002819050.1 XP_003777624.1 Gorilla gorilla XP_004046943.1 NP_001028241.1 Homo sapiens NP_004086.1 NP_034254.1 Macaca mulatta EHH28418.1 XP_007638835.1 Ophiophagus hannah ETE67930 ETE71675.1