SIRT6 regulates Ras-related protein R-Ras2 by lysine defatty-acylation
- Cornell University, United States
- Howard Hughes Medical Institute, Cornell University, United States
Figures
Figure 1 with 1 supplement
Identification of defatty-acylation targets of SIRT6 that contribute to its function in cell proliferation.
(A) Cell proliferation of Sirt6 KO MEFs stably expressing pCDH empty vector, SIRT6 WT, G60A, or H133Y. Cell proliferation was assayed and quantified using crystal violet staining. Values with error …
Figure 1—figure supplement 1
Schematic overview of the SILAC experiment to compare the protein abundance in Sirt6 WT and KO MEFs.
https://doi.org/10.7554/eLife.25158.004
Figure 2 with 4 supplements
Validation of R-Ras2 as the defatty-acylation target of SIRT6.
(A) mRNA and protein levels of R-Ras2 in Sirt6 WT and KO MEFs. (B) In-gel fluorescence (with NH2OH treatment) showing that R-Ras2 has higher lysine fatty acylation level in Sirt6 KO MEFs than in Sirt…
Figure 2—figure supplement 1
Scheme showing the in-gel fluorescence method with Alk14 metabolic labeling to identify R-Ras2 as a lysine fatty acylated protein.
FLAG-tagged R-Ras2 protein was enriched from whole cell lysates by FlAG immunoprecipitation. Alk14-labeled R-Ras2 protein was detected by in-gel fluorescence after incorporating BODIPY (B)-azide …
Figure 2—figure supplement 2
Validation of R-Ras2 as the defatty-acylation target of SIRT6.
(A) Full gel image of R-Ras2 fatty acylation level with or without NH2OH treatment in Sirt6 WT and KO MEFs. (B) Lysine fatty acylation levels of overexpressed R-Ras2 in Sirt6 WT and KO MEFs. (C) …
Figure 2—figure supplement 3
Total ion chromatogram (TIC), extracted ion chromatogram (XIC) and parent MS (MS1) of Ak14 modified R-Ras2 peptide.
https://doi.org/10.7554/eLife.25158.008
Figure 2—figure supplement 4
Lysine fatty acylation targets R-Ras2 to plasma membrane.
(A) Confocal imaging showing subcellular localization of GFP-tagged R-Ras2 WT and 4KR in Sirt6 WT and KO MEFs. (B) Subcellular fractionation of R-Ras2 WT and 4KR in HEK 293T cells with palmitic acid …
Figure 3 with 1 supplement
SIRT6 defatty-acylase activity is required for regulating R-Ras2 lysine fatty acylation and subcellular localization.
(A) Detection of R-Ras2 lysine fatty acylation levels in Sirt6 KO MEFs expressing pCDH empty vector, SIRT6 WT, G60A or H133Y by in-gel fluorescence. (B) In-gel fluorescence showed that SIRT6 WT and …
Figure 3—figure supplement 1
The defatty-acylase activity of SIRT6 regulates R-Ras2 subcellular localization.
(A) Confocal imaging showing the localization of GFP-tagged R-Ras2 in Sirt6 KO MEFs expressing pCDH empty vector, SIRT6 WT, G60A or H133Y. (B) Subcellular fractionation showing the localization of …
Figure 4 with 2 supplements
Lysine fatty acylation of R-Ras2 activates PI3K-Akt pathway and promotes cell proliferation in MEFs.
(A) Co-IP experiment showed that R-Ras2 WT in Sirt6 KO MEFs had more p110α interaction than R-Ras2 WT in Sirt6 WT MEFs and R-Ras2 4KR in Sirt6 WT or KO MEFs. (B) Knockdown of R-Ras2 in Sirt6 KO MEFs …
Figure 4—figure supplement 1
Raf-RBD binding assay showed that both overexpressed and endogenous R-Ras2 did not bind Raf RBD-conjugated argarose beads.
Total endogenous Ras (H-Ras, N-Ras and K-Ras) was used as the positive control.
Figure 4—figure supplement 2
The images of other western blots for quantification.
(A) Knockdown of R-Ras2 in Sirt6 KO MEFs decreased p-Akt Thr308, but not p-Akt Ser473, to a level similar to that in Sirt6 WT MEFs. (B) Expressing SIRT6 WT or SIRT6 G60A in Sirt6 KO MEFs decreased …
Additional files
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Supplementary file 1
Protein list of Alk12 SILAC in Sirt6 WT and KO MEFs
- https://doi.org/10.7554/eLife.25158.015
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Supplementary file 2
Protein list of Alk14 SILAC in Sirt6 WT and KO MEFs
- https://doi.org/10.7554/eLife.25158.016
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Supplementary file 3
Protein list of total protein SILAC in Sirt6 WT and KO MEFs
- https://doi.org/10.7554/eLife.25158.017
