ABSTRACT
The E3 ligase ARIH2 has an unusual structure and mechanism of elongating ubiquitin chains. To understand its physiological role, we generated gene-targeted mice deficient in ARIH2. ARIH2 deficiency resulted in the embryonic death of C57BL/6 mice. On a mixed genetic background, the lethality was attenuated, with some mice surviving beyond weaning and then succumbing to an aggressive multiorgan inflammatory response. We found that in dendritic cells (DCs), ARIH2 caused degradation of the inhibitor IκBß in the nucleus, which abrogated its ability to sequester, protect and transcriptionally coactivate the transcription factor subunit p65 in the nucleus. Loss of ARIH2 caused dysregulated activation of the transcription factor NF-κB in DCs, which led to lethal activation of the immune system in ARIH2-sufficent mice reconstituted with ARIH2-deficient hematopoietic stem cells. Our data have therapeutic implications for targeting ARIH2 function.
Subject(s)
Dendritic Cells/immunology , Embryonic Development/immunology , Multiple Organ Failure/immunology , Ubiquitin-Protein Ligases/physiology , Animals , Cells, Cultured , Disease Models, Animal , Embryonic Development/genetics , Hematopoiesis/genetics , Humans , Immune System/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Targeted Therapy , Multiple Organ Failure/genetics , NF-kappa B/metabolism , Transcriptional Activation/immunology , Ubiquitin-Protein Ligases/genetics , Ubiquitination/genetics , Ubiquitination/immunologyABSTRACT
MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance.
Subject(s)
Codon, Initiator , Immunologic Surveillance/genetics , Peptides/immunology , Protein Biosynthesis/immunology , Animals , Histocompatibility Antigens Class I , Humans , Peptide Chain Initiation, Translational , Peptidyl Transferases/antagonists & inhibitors , RNA, Messenger , Ribosomes/geneticsABSTRACT
Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.