Fig. 4: T203 phosphorylation is necessary for TRIM37 to stabilize PARP1 by mono-ubiquitination of K337. | Nature Communications

Fig. 4: T203 phosphorylation is necessary for TRIM37 to stabilize PARP1 by mono-ubiquitination of K337.

From: TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation

Fig. 4: T203 phosphorylation is necessary for TRIM37 to stabilize PARP1 by mono-ubiquitination of K337.The alternative text for this image may have been generated using AI.

a Immunoprecipitation of PARP1 and the identification of phosphorylated proteins by tandem mass spectrometry in early MSCs. Interpretation of MS2 spectra revealed enriched phosphopeptides associated with PARP1. Phosphor-TRIM37T203 is indicated. Detailed information is in Supplementary Table 3. b Co-immunoprecipitation assay showed that the exogenous TRIM37 binds to PARP1 in 293 T cells cotransfected with the plasmids of Flag-TRIM37 and HA-PARP1. c Western blotting for the protein levels of immunoprecipitated HA-PARP1 in 293 T cells cotransfected with plasmids of Flag-TRIM37, His-ubiqutin (His-Ub), and HA-PARP1/wild-type (WT) or indicated mutants. The overexpression of HA-PARP1 with K337R mutation, rather than with K438R mutation, significantly reduced the immunoprecipitated HA-PARP1 and His-Ub protein levels compared to WT HA-PARP1. d, e Western blotting for the expression of Flag-TRIM37 bound to immunoprecipitated PHA-ARP1 in 293 T cells cotransfected with plasmids of HA-PARP1, His-Ub, and Flag-TRIM37/WT or indicated mutant. d The overexpression of Flag-TRIM37 with T203A mutation reduced Flag-TRIM37 binding to and ubiquitination of HA-PARP1 compared to WT Flag-TRIM37. eThe overexpression of Flag-TRIM37 with T203E mutation increased Flag-TRIM37 bind–ing to, and the ubiquitination and stabilization of HA-PARP1 compared to WT Flag-TRIM37. f–h Early MSCs were lentivirally transduced with scrambled (CTR) and TRIM37 shRNAs (TRIM37-KD #1 & #2), followed by (f) western blotting for the detection of PARP1, DNMT1, and senescence marker levels, (g) WST-1 assay for growth curve analysis, and (h) induction for osteogenic and adipogenic differentiation for 14 d. TRIM37 knockdown decreased the PARP1 and DNMT1 full-length protein levels, increased the senescence marker levels, inhibited the cell proliferation, and suppressed the differentiation of cells into osteoblasts and adipocytes. Results are expressed as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ****p < 0.0001; ordinary one-way or two-way ANOVA). Exact p-values are shown in the Source Data file. Scale bar: 500 μm.

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