Akt+ IKKα/β+ Rab5+ Signalosome Mediate the Endosomal Recruitment of Sec61 and Contribute to Cross-Presentation in Bone Marrow Precursor Cells

Abstract

Cross-presentation in dendritic cells (DC) requires the endosomal relocations of internalized antigens and the endoplasmic reticulum protein Sec61. Despite the fact that endotoxin-containing pathogen and endotoxin-free antigen have different effects on protein kinase B (Akt) and I-kappa B Kinase α/β (IKKα/β) activation, the exact roles of Akt phosphorylation, IKKα or IKKβ activation in endotoxin-containing pathogen-derived cross-presentation are poorly understood. In this study, endotoxin-free ovalbumin supplemented with endotoxin was used as a model pathogen. We investigated the effects of endotoxin-containing pathogen and endotoxin-free antigen on Akt phosphorylation, IKKα/β activation, and explored the mechanisms that the endotoxin-containing pathogen orchestrating the endosomal recruitment of Sec61 of the cross-presentation in bone marrow precursor cells (BMPC). We demonstrated that endotoxin-containing pathogen and endotoxin-free antigen efficiently induced the phosphorylation of Akt-IKKα/β and Akt-IKKα, respectively. Endotoxin-containing pathogen derived Akt+ IKKα/β+ Rab5+ signalosome, together with augmented the recruitment of Sec61 toward endosome, lead to the increased cross-presentation in BMPC. Importantly, the endosomal recruitment of Sec61 was partly mediated by the formation of Akt+ IKKα/β+ signalosome. Thus, these data suggest that Akt+ IKKα/β+ Rab5+ signalosome contribute to endotoxin-containing pathogen-induced the endosomal recruitment of Sec61 and the superior efficacy of cross-presentation in BMPC.

K48-Linked Ubiquitination Contributes to Nicotine-Augmented Bone Marrow-Derived Dendritic-Cell-Mediated Adaptive Immunity

“…siRNA transfection was performed according to the guideline of the manufacturer and our previous description [ 19 ]. Briefly, buffer A and B were respectively prepared by diluting 2–8 µL of siRNA duplex (20–80 pmols siRNA) or transfection reagent (sc-29528) into 100 µL siRNA transfection medium (sc-36868).…”

“…Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP) was performed according to previous description [ 19 ]. Briefly, BM-DC stimulated with nicotine was harvested in the presence of MG132 (20 × 10 −6 mol/L) and lysed in the buffer of Pierce™ Co-Immunoprecipitation Kit (Cat.…”

“…Western blot analysis was performed according to previous description [ 19 ]. Briefly, the cellular proteins were extracted and loaded onto 6~8% SDS-PAGE.…”

“…During this process, p97, an AAA-ATPase located in the endoplasmic reticulum (ER), relocates toward the endosomes and provides the driving force for the transport of misfolded proteins [ 15 , 16 ]. Sec61, another ER component protein, was also documented to relocate from the ER during DC cross-presentation [ 17 , 18 , 19 ]. Although protein kinase B (Akt) + I-kappa B Kinase α/β (IKKα/β) + Rab5 + signalosome is essential for the recruitment of Sec61 toward endosome [ 19 ], the ubiquitination of mannose receptor (MR) was also essential for the endosomal relocation of p97 [ 18 ].…”

“…Sec61, another ER component protein, was also documented to relocate from the ER during DC cross-presentation [ 17 , 18 , 19 ]. Although protein kinase B (Akt) + I-kappa B Kinase α/β (IKKα/β) + Rab5 + signalosome is essential for the recruitment of Sec61 toward endosome [ 19 ], the ubiquitination of mannose receptor (MR) was also essential for the endosomal relocation of p97 [ 18 ]. In our previous studies, the activation of nuclear factor kappa-B (NF-κB) was found to recruit the endosomal relocation of transporter associated with antigen processing (TAP) [ 20 , 21 ].…”