TNFR-associated factor (TRAF)6 is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. In this study, we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4⁺ T cells conjugated with staphylococcal enterotoxin E–pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference–induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine phosphorylation, and association with tyrosine kinase ZAP70. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lys⁸⁸ by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR–LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, indicating that LAT also plays an adapter role in TCR/CD28-induced activation of TRAF6.

Monocyte/macrophage differentiation represents a major branch of hematopoiesis and is a central event in the immune response, but the molecular mechanisms underlying are not fully delineated. Here we show that p38 mitogen-activated protein kinase (MAPK) interacting protein (p38IP) is downregulated during monocyte/macrophage differentiation in vitro. Overexpression of p38IP halted monocyte/macrophage differentiation, whereas forward knockdown of p38IP by RNA interference induced G1/S arrest and promoted monocyte differentiation into macrophages and the maturation of macrophages as well. Moreover, we found that miR-200b-3p was upregulated during monocyte/macrophage differentiation and mediated the downregulation of p38IP by binding to the 3′ untranslated terminal region of p38IP mRNA. Overexpression of a miR-200b-3p mimic resembled the effect of p38IP knockdown, whereas a miR-200b-3p inhibitor blocked monocyte/macrophage differentiation by enhancing p38IP expression. Further western blotting analysis revealed that p38IP downregulation enhanced the activity of p38 MAPK and the subsequent accumulation of cyclin-dependent kinase inhibitor p21, thus promoting G1/S arrest and monocyte/macrophage differentiation. Moreover, the decline of GCN5 acetyltransferase caused by p38IP downregulation was required but was not sufficient for monocyte/macrophage differentiation. This study demonstrated a new role for p38IP and a novel miR-200b-3p/p38IP pair in the regulation of monocyte/macrophage differentiation.

P38-interacting protein (p38IP) is a component of the GCN5 histone acetyltransferase-containing coactivator complex (GCN5-SAGA complex). It remains unclear whether p38IP or GCN5-SAGA is involved in cell cycle regulation. Using RNA interference to knock down p38IP, we observed that cells were arrested at the G₂/M phase, exhibiting accumulation of cyclins, shrunken spindles, and hypoacetylation of α-tubulin. Further analysis revealed that knockdown of p38IP led to proteasome-dependent degradation of GCN5. GCN5 associated with and acetylated α-tubulin, and recovering GCN5 protein levels in p38IP knockdown cells by ectopic expression of GCN5 efficiently reversed α-tubulin hypoacetylation and G₂/M arrest. During the G₂/M transition, the association of α-tubulin with GCN5 increased, and the acetylation of α-tubulin reached a peak. Biochemical analyses demonstrated that the interaction between p38IP and GCN5 depended on the p38IP N terminus (1–381 amino acids) and GCN5 histone acetyltransferase domain and bromodomain. The p38IP N terminus could effectively reverse p38IP depletion-induced GCN5 degradation, thus recovering α-tubulin acetylation and G₂/M progression. p38IP-mediated suppression of GCN5 ubiquitination most likely occurs via nuclear sequestration of GCN5. Our data indicate that the GCN5-SAGA complex is required for G₂/M progression, mainly because p38IP promotes the acetylation of α-tubulin by preventing the degradation of GCN5, in turn facilitating the formation of the mitotic spindle.

Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the worst outcome. CMS4 CRC is notoriously resistant against therapeutic interventions, as demonstrated by preclinical studies and retrospective clinical observations. Here, we report the finding that two clinically employed agents, everolimus (EVE) and plicamycin (PLI), efficiently target the prototypic CMS4 cell line MDST8. As compared to the prototypic CMS1 cell line LoVo, MDST8 cells treated with EVE or PLI demonstrated stronger cytostatic and cytotoxic effects, increased signs of apoptosis and autophagy, as well as a more pronounced inhibition of DNA-to-RNA transcription and RNA-to-protein translation. Moreover, nontoxic doses of EVE and PLI induced the shrinkage of MDST8 tumors in mice, yet had only minor tumor growth-reducing effects on LoVo tumors. Altogether, these results suggest that EVE and PLI should be evaluated for their clinical activity against CMS4 CRC.

The nuclear pore complex (NPC) is the sole and selective gateway for nuclear transport, and its dysfunction has been associated with many diseases. The metazoan NPC subcomplex RanBP2, which consists of RanBP2 (Nup358), RanGAP1-SUMO1, and Ubc9, regulates the assembly and function of the NPC. The roles of immune signaling in regulation of NPC remain poorly understood. Here, we show that in human and murine T cells, following T-cell receptor (TCR) stimulation, protein kinase C-θ (PKC-θ) directly phosphorylates RanGAP1 to facilitate RanBP2 subcomplex assembly and nuclear import and, thus, the nuclear translocation of AP-1 transcription factor. Mechanistically, TCR stimulation induces the translocation of activated PKC-θ to the NPC, where it interacts with and phosphorylates RanGAP1 on Ser⁵⁰⁴ and Ser⁵⁰⁶. RanGAP1 phosphorylation increases its binding affinity for Ubc9, thereby promoting sumoylation of RanGAP1 and, finally, assembly of the RanBP2 subcomplex. Our findings reveal an unexpected role of PKC-θ as a direct regulator of nuclear import and uncover a phosphorylation-dependent sumoylation of RanGAP1, delineating a novel link between TCR signaling and assembly of the RanBP2 NPC subcomplex.