Fig. 7: NAT10-mediated acetylation stabilizes RACK1 via suppressing its ubiquitination and degradation.

a Naïve CD4⁺ T cells isolated from NAT10^WT and NAT10^cKO mice were activated with αCD3 plus αCD28 for 24 h. The protein abundance of RACK1 was measured by IB. b WT and NAT10-deficient naïve CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 for 22 h, and treated with 20 µM MG132 for another 2 h. The cell lysates were subjected to IB to measure protein abundance of RACK1. c, d IB analysis of ubiquitinated RACK1 (detected with anti-ubiquitin) among proteins IP with anti-HA (RACK1) from lysates of HEK293T cells transfected with FLAG-tagged wild-type ubiquitin (c) or mutant ubiquitin (d) in the presence (+) or absence (−) of expression plasmids for Myc-tagged NAT10 and HA-tagged RACK1 (top), and IB analysis of total cellular polyubiquitination (FLAG-Ub), RACK1 and TUBLIN in lysates without immunoprecipitation (below). e IB analyses of αCD3 + αCD28-triggered K48-linked ubiquitination of RACK1 in WT and NAT10-deficient CD4⁺ T cells. RACK1 was isolated by IP and followed by IB detection of ubiquitin. f Schematic diagram showing identified ubiquitinated lysine sites of RACK1 with or without NAT10 co-expression by mass spectrum. “-NAT10 or +NAT10” means “with or without NAT10 overexpression”. g Venn plot showing the same site of amino acid which was both detected acetylation and ubiquitination. h IB analysis of various ubiquitinated mutant RACK1 among proteins IP with HA from lysates of HEK293T cells co-transfected with Myc-tagged wild-type ubiquitin (top), and immunoblot analysis of total cellular polyubiquitination (Myc-Ub) and RACK1. i K175, K185 and K271 ubiquitination of RACK1 in activated T cells identified by mass spectrometry. j The acetylation of Strep-RACK1 mediated by recombinant FLAG-NAT10 was measured by IB. k mCherry⁺ WT and RACK1 K185Q mutant-transfected NAT10-deficient CD4⁺ T cells isolated by FACS, and treated with 20 µM MG132 for 2 h. The protein level of WT and RACK1 K185Q mutant were detected by IB. l The acetylation of Strep-RACK1 mediated by recombinant WT FLAG-NAT10 and NAT10 G641E mutant was measured by IB. m HEK293T cells were transfected with Myc-tagged ubiquitin, HA-tagged RACK1 and FLAG-NAT10 G641 mutant and its WT control, and subjected to ubiquitination assay 36 h later. n, o mCherry⁺ WT and RACK1 K185Q mutant-transfected naïve NAT10-deficient CD4⁺ T cells were labeled with CFSE, activated with anti-CD3/anti-CD28 (n = 4). The proliferation of these cells was measured with CFSE dilution by FACS and after 48 h and presented as bar graph. p–q ECAR and OCR of mCherry⁺ WT and RACK1 K185Q mutant-transfected naïve NAT10-deficient CD4⁺ T cells stimulated with anti-CD3 plus anti-CD28 for 18 h were measured by Agilent Seahorse XF Analyzers (n = 3). r, s mCherry⁺ WT and RACK1 K185Q mutant-transfected naïve NAT10-deficient CD4⁺ T cells were polarized under Th1 differentiation condition (n = 3). The expression of IFN-γ were analyzed by FACS after 96 h. Source data are provided as a Source Data file. Data were represented as mean ± S.D. Three independent experiments at least were performed in data represented above. Statistical significance was determined by one-way ANOVA with multiple comparisons (o, s). *P < 0.05, **P < 0.01, ***P < 0.005, ns no significant.