Abstract
Many intracellular proteins are modified by N-acetylglucosamine, a post-translational modification termed O-GlcNAc. This modification is found on serine and threonine side chains and has the potential to regulate signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. We find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P), culminating in the phosphorylation of myosin light chain (MLC) and cellular contraction. Specifically, O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen-matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing.
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Data availability
RT-PCR primers were designed using the MIT Primer3 design database (http://biotools.nubic.northwestern.edu/Primer3.html) with sequences found using the NCBI Gene Search tool (ncbi.nlm.nih.gov/gene). Source data are provided with this paper.
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Acknowledgements
We thank F. Grinnell for consultations on collagen matrix contraction. This research was supported by the American Cancer Society Research Scholar Grant (RSG-14-225-01-CCG), the University of Southern California, the Anton Burg Foundation and the National Institutes of Health (R01GM125939; to M.R.P.). N.J.P. is supported by NIGMS T32GM118289. We thank K. Moremen at the University of Georgia, who is supported by the National Institutes of Health (P41GM103390 and R01GM130915), for the generous gift of GalT(Y289L).
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N.J.P., A.R.B., N.D. and M.R.P. designed experiments and interpreted data. N.J.P. and A.R.B. carried out cellular phenotype and western blotting experiments. N.J.P. generated the MYPT1 stable cell lines and performed the associated experiments. N.J.P. also performed the co-IP, apoptosis and collagen matrix experiments. N.D. performed the analysis of MYPT1 O-GlcNAc levels and dynamics. N.J.P., A.R.B. and M.R.P. prepared the manuscript.
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Extended data
Extended Data Figure 1 Modulating O-GlcNAcylation levels and identification of S1P.
a, Schematic layout of our general experimental conditions. b, O-GlcNAc levels can be changed upon OGT or OGA inhibition. NIH3T3 cells were treated with either the OGT inhibitor 5SGlcNAc, the OGA inhibitor Thiamet G, or DMSO vehicle respectively before the O-GlcNAc levels were analyzed by Western blotting. c, Only serum- or S1P-treatment results in notable cell contraction when O-GlcNAc levels have been lowered. NIH3T3 cells were treated with DMSO or 5SGlcNAc. The indicated signaling molecules were then added, and the contraction phenotype was then visualized using bright-field microscopy. The data in b-c is representative of 2 biological replicates.
Extended Data Figure 2 O-GlcNAc controls the sensitivity of fibroblasts to sphingosine-1-phosphate (S1P) mediated cell contraction.
a, O-GlcNAc levels can be changed upon OGT inhibition. NIH3T3 cells were treated with either the OGT inhibitor ST060266 or DMSO vehicle respectively before the O-GlcNAc levels were analyzed by Western blotting. b, O-GlcNAc levels can be lowered by OGT RNAi. NIH3T3 cells were transfected with RNAi targeting OGT or a scrambled sequence before analysis by Western blotting. c, NIH3T3 cells were treated with the indicated combinations of DMSO, the OGT inhibitor ST060266 (200 μM) and/or S1P. The contraction phenotype was then visualized using bright-field microscopy. d, NIH3T3 cells were transfected with either scramble or OGT-targeted RNAi for 48 h before addition of either DMSO or S1P. The contraction phenotype was then visualized using bright-field microscopy. e, O-GlcNAc levels can be changed by culturing cells in different glucose concentrations. NIH3T3 cells were cultured in the indicated concentrations of glucose before analysis by Western blotting. The data in a-e is representative of at least 2 biological replicates.
Extended Data Figure 3 Signaling through the second S1P receptor, S1PR2, is responsible for the contraction phenotype.
a, NIH3T3 cells can express all five S1P GPCRs (S1PR1 to 5). mRNA was collected from NIH3T3 cells before being subjected to RT-PCR and visualization on an DNA-agarose gel. These data are representative of 2 biological replicates. b, Antagonizing S1PR2, but not the other receptors, inhibits S1P-mediated cell contraction. NIH3T3 cells were treated with either DMSO or 5SGlcNAc. The same cells were then treated with either additional DMSO or the indicated selective antagonists followed by S1P. The contraction phenotype was then visualized using bright-field microscopy. c, Quantitation of the data in (b). Results are the mean ± SEM of the relative culture plate area taken-up by cells in at least three randomly selected frames. Statistical significance was determined using a 2-tailed student’s t-test. d, S1PR5 agonism does not induce cell contraction. NIH3T3 cells were treated with either DMSO or 5SGlcNAc. The same cells were then treated with either S1P or the S1PR5-selective agonist A971432. The contraction phenotype was then visualized using bright-field microscopy. e, Quantitation of the data in (d). Results are the mean ± SEM of the relative culture plate area taken-up by cells in at least three randomly selected frames. Statistical significance was determined using a 2-tailed student’s t-test. f, Lowering O-GlcNAc levels increases the sensitivity of NIH3T3 cells to S1PR2 induced cell contraction. NIH3T3 cells were treated with either DMSO or 5SGlcNAc before addition of the indicated concentrations of the S1PR2-selective agonist CYM5220. The contraction phenotype was then visualized using bright-field microscopy. g, Quantitation of the data in (f). Results are the mean ± SEM of the relative culture plate area taken-up by cells in four randomly selected frames. Statistical significance was determined using a 2-way ANOVA test followed by Sidak’s multiple comparisons test. h, S1PR2 knockdown using siRNA blocks contraction phenotype. NIH3T3 cells were transfected with either scramble or S1Pr2-targeted RNAi before addition of DMSO or S1P. The contraction phenotype was then visualized using bright-field microscopy. i, Quantitation of the data in (h). Results are the mean ± SEM of the relative culture plate area taken-up by cells in three randomly selected frames. Statistical significance was determined using a 2-way ANOVA test followed by Sidak’s multiple comparisons test.
Extended Data Figure 4 Inhibition of Rho kinase (ROCK1/2) blocks S1P-mediated cell contraction.
NIH3T3 cells were treated with either DMSO or 5SGlcNAc. The same cells were then treated with either additional DMSO or the ROCK1/2 inhibitor Y27632 followed by the indicated concentrations of S1P. The contraction phenotype was then visualized using bright-field microscopy. The results were then quantified and are presented as mean ± SEM of the relative culture plate area taken-up by cells in four randomly selected frames.
Extended Data Figure 5 Analysis of MYPT1 mutants shows that deletion of the serine/threonine domain sensitizes cells to S1P-mediated contraction.
a, Sequence alignment of MYPT1 and the different mutants tested here. Red indicates potential O-GlcNAc modification sites (S or T), and blue indicates O-GlcNAc sites previously identified by mass spectrometry. MYTP1(S/TtoA) mutates all of the previously identified O-GlcNAc sites, MYPT1(d564-578) deletes the first serine/threonine rich region, MYPT1(d588-602) deletes the second serine/threonine rich region, and MYPT1Δ deletes the entire serine/threonine rich domain. b, MYPT1Δ, and to a lesser extent MYPT1(d564-578), sensitizes cells to S1P-mediated contraction. NIH3T3 cells expressing the indicated MYPT1 proteins and the endogenous copy was removed by RNAi. DMSO or S1P was then added and the contraction of the cells was measured. Results are the mean ± SEM of the relative culture plate area taken-up by cells in four randomly selected frames. Statistical significance was determined using a 2-way ANOVA test followed by Sidak’s multiple comparisons test.
Extended Data Figure 6 Deletion of the MYPT1 serine/threonine rich domain yields MYPT1Δ with reduced O-GlcNAc modification.
a, Schematic of the MYPT1Δ protein, which lacks the major O-GlcNAc region of the protein. b&c) MYPT1Δ loses a notable amount of O-GlcNAc. The O-GlcNAc levels of FLAG-tagged MYPT1 or MYPT1Δ were analyzed using mass shifting or IP-Western blot. The data in are representative of at least 2 biological replicates.
Extended Data Figure 7 MYPT1Δ is an active phosphatase that can dephosphorylate MLC and return cells to a relaxed state.
a, If MYPT1Δ is an active phosphatase, we expect that it will become phosphorylated and deactivated by ROCK after S1P treatment but will return to a desphosphorylated and active state after a longer period of time. This will result in MLC dephosphorylation and relaxation of the cells. This is exactly what we observed by Western blotting. These data are representative of 2 biological replicates. b, Cells expressing MYPT1Δ return to a relaxed state over 180 min. The contraction phenotype was visualized using bright-field microscopy. The results were then quantified and are presented as mean ± SEM of the relative culture plate area taken-up by cells in four randomly selected frames. Statistical significance was determined using a 2-way ANOVA test followed by Sidak’s multiple comparisons test.
Extended Data Figure 8 O-GlcNAc blocks the interaction between MYPT1 and ROCK.
NIH3T3 cells expressing flag-tagged MYPT1 were treated with either DMSO or 5SGlcNAc (200 μM). An anti-flag co-immunoprecipitation was then performed using the Catch and Release system (Thermo). ROCK enrichment was detected by Western blotting and normalized to overall protein capture (Coomassie staining). The results were quantities and presented as mean ± SEM of the normalized ROCK levels (n = 3 biological replicates). Statistical significance was determined using a 2-tailed, unpaired Student’s t-test.
Extended Data Figure 9 Direct MYPT1 O-GlcNAcylation is largely responsible for the phenotype.
a, NIH3T3 cells stably expressing either MYPT1 or MYTP1Δ were transfected with RNAi to downregulate endogenous MYTP1. MYTP1-expressing cells were then treated with either DMSO or 5SGlcNAc. Cells under all three sets of conditions were then treated with the indicated concentrations of S1P. The contraction phenotype was visualized using bright-field microscopy. The results were then quantified and are presented as mean ± SEM of the relative culture plate area taken-up by cells in four randomly selected frames. Statistical significance was determined using a 2-way ANOVA test followed by Sidak’s multiple comparisons test. b, NIH3T3 cells stably expressing either MYPT1 or MYTP1Δ were transfected with RNAi to downregulate endogenous MYTP1. MYTP1-expressing cells were then treated with either DMSO or Thiamet-G. Cells under all three sets of conditions were then treated with the indicated concentrations of S1P. The contraction phenotype was visualized using bright-field microscopy. The results were then quantified and are presented as mean ± SEM of the relative culture plate area taken-up by cells in four randomly selected frames. Statistical significance was determined using a 2-way ANOVA test followed by Sidak’s multiple comparisons test.
Extended Data Figure 10 Our experimental model.
a, MYPT1 O-GlcNAcylation inhibits its phosphorylation by ROCK1/2. This maintains MYPT1 phosphatase activity, resulting in inactive MLC and no actin contraction. Loss of O-GlcNAc enables ROCK1/2 to phosphorylate and deactivate MYTP1. b, Therefore, MYPT1 O-GlcNAcylation levels control the sensitivity of cells to the concentration of S1P, where more MYTP1 O-GlcNAcylation requires more S1P to illicit actin contraction and cell detachment.
Supplementary information
Supplementary Information
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Supplementary Video 1
O-GlcNAc levels control the contraction of fibroblasts in response to serum.
Supplementary Video 2
O-GlcNAc levels control the contraction of fibroblasts in response to S1P.
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Pedowitz, N.J., Batt, A.R., Darabedian, N. et al. MYPT1 O-GlcNAc modification regulates sphingosine-1-phosphate mediated contraction. Nat Chem Biol 17, 169–177 (2021). https://doi.org/10.1038/s41589-020-0640-8
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DOI: https://doi.org/10.1038/s41589-020-0640-8
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