Fig. 2: TRIM23 is phosphorylated at Ser-39.

a Schematic representation of the domain organization of TRIM23 with the phospho-S39 (pS39) residue illustrated. RING, Really Interesting New Gene domain; BB1/2, B-Box 1 and 2; CC, coiled-coil domain; ARF, ADP-ribosylation factor domain. b Amino acid sequence of the region containing the S39 residue in TRIM23 from the indicated species. Numbers indicate amino acid positions. c Tandem mass spectra of the tryptic peptide K.VLECGVCEDVFpS₃₉LQGDKVPR.L of TRIM23-FLAG affinity-purified from transiently transfected HEK293T cells that identified phosphorylation at S39. d Phosphorylation of TRIM23-FLAG WT and S39A mutant in transiently transfected HEK293T cells that were either mock-treated (–) or treated with calyculin A (Cal A, a phosphatase inhibitor), determined at 48 h post-transfection by immunoprecipitation (IP) with anti-FLAG and IB with anti-p-S39-TRIM23 antibody. e S39 phosphorylation and poly-ubiquitination of FLAG-TRIM23 stably expressed in HDFs that were infected with mutHSV-1 as indicated, determined at 8 h post-infection by IP with anti-FLAG and IB with anti-p-S39-TRIM23 or anti-ubiquitin (Ub). f S39 phosphorylation of endogenous TRIM23 in HDFs that were either mock-treated or infected with WT HSV-1 or mutHSV-1 (MOI 5 for both) for 16 h, determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of p-S39-TRIM23-positive cells for the data shown in (f). h Time course analysis of endogenous TRIM23 S39 phosphorylation in HDFs that were either mock-treated or infected with mutHSV-1 (MOI 5) for the indicated times, determined as in (f). DAPI, nuclei (blue). Scale bar, 20 µm. i Quantification of p-S39-TRIM23-positive cells for the data shown in (h). j Upper: Ribbon representation of the crystal structure of the TRIM23-RING dimer (green/blue) in complex with Ub-loaded E2 (orange/red)⁴³. The loops containing S39 are highlighted in pink. Lower: Close-up view of the RING S39 loop, stabilized in part by a bifurcated hydrogen bond (yellow), and its proximity to the Ub or E2 binding interface. k K27-linked auto-polyubiquitination of FLAG-tagged TRIM23 WT or mutants in transiently transfected HEK293T cells that co-expressed an HA-tagged ubiquitin mutant (HA-Ub-K27only), determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-HA. Data in (c) are from one unbiased MS analysis, data in (d–i and k) are representative of at least two independent experiments (mean ± SD of n  = 4 biological replicates (g, i). ****p < 0.0001 (two-tailed Student’s t test (g, i). Source data are provided as a Source Data file.