Fig. 4: β-AR-PKA phosphorylation of MYPT1 inhibits MYPT1-PP1β activity. | Nature Communications

Fig. 4: β-AR-PKA phosphorylation of MYPT1 inhibits MYPT1-PP1β activity.

From: MYPT1-PP1β phosphatase negatively regulates both chromatin landscape and co-activator recruitment for beige adipogenesis

Fig. 4: β-AR-PKA phosphorylation of MYPT1 inhibits MYPT1-PP1β activity.The alternative text for this image may have been generated using AI.

a Volcano plot of ISO/Vehicle phosphoproteome in im-scWAT pre-treated with ISO (10 μM) for 40 min on day 0 (Isobaric Tag Labeling) (the same samples were processed and subjected to LC-MS/MS analysis for three times as technical replicates to verify measurement accuracy). X-axis, log2 FC of reporter ion intensity: Y-axis, −log10 P-value of reporter ion intensity change. The number of total phosphopeptides (n) and phosphopeptides with increased (FC > 1.5, P < 0.05) and decreased (FC < 1/1.5, P < 0.05) phosphorylation levels by MYPT1 depletion are shown. b Post-translational modifications of MYPT1 identified by phosphoproteomics in (a). MS/MS spectrum of P-MYPT1 fragment from R692 to K707. c TMT reporter ion intensities derived from MS/MS spectrum of P-MYPT1 fragment. Data are mean ± SEM of three technical replicates. P-values by paired two-tailed t test. d PKA consensus site is conserved among various species. e P-MYPT1 and MYPT1 immunoblotting from im-scWAT pre-cultured in 0.1% bovine serum albumin containing DMEM for 3 h and treated with ISO (10 μM) for 0, 5, 10, and 20 min on day 0 (f) P-MYPT1 and MYPT1 immunoblotting from im-scWAT pre-cultured in 0.1% bovine serum albumin containing DMEM for 3 h and treated with H89 (20 μM) for 20 min and then treated with ISO (10 μM) for 10 min on day 0. g P-JMJD1A and JMJD1A immunoblotting following immunoprecipitation with anti-mouse JMJD1A from WT- or T696A-human MYPT1-transduced im-scWAT treated with ISO (10 μM) for 0, 20, and 40 min on day 0 transfected with si-Mypt1 #3. h Thermogenic gene mRNA expression in day 8 im-scWAT differentiated for beige adipogenesis transduced with WT- or T696A-human-MYPT1 transfected with si-Mypt1 #3. Data are mean ± SEM of four biological replicates. P-values by two-tailed Student’s t test. i PKA is activated upon β-AR activation, while MYPT1-PP1β is inactivated via Thr694 phosphorylation of MYPT1 by PKA, enabling efficient phosphorylation of JMJD1A in response to cold stress. e, f, g Representative of three independent experiments. Source data are provided as a Source data file.

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