ABSTRACT
The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein-protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs.
Subject(s)
Cell Nucleus Structures/metabolism , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Alternative Splicing , HEK293 Cells , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Nuclear Proteins/genetics , Promyelocytic Leukemia Protein , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Two-Hybrid System TechniquesABSTRACT
Deletion mutants of Aeromonas salmonicida subsp. salmonicida were used to determine the effect of the type three secretion system (TTSS) on Atlantic salmon anterior head kidney leucocytes (AHKL). One strain had a deletion in the outer membrane pore gene, ascC; and the other in three effector genes: aopO, aopH and aexT (we call this strain Deltaaop3). Host cell invasion success and 24h survival were depressed in DeltaascC, as was 24h survival of Deltaaop3, when compared to the wild type strain. Challenge of AHKLs with A449 or TTSS mutants stimulated expression of the inflammatory mediators IL-8, IL-1 and TNFalpha at two bacterial concentrations (A(600) 0.1, 0.01). Expression of IL-12 was not stimulated in DeltaascC challenged cells, whereas A449 and Deltaaop3 challenge resulted in an up-regulation of IL-12 in AHKLs, 2- and 4-fold higher than PBS, respectively. Only the wild type strain elicited a significant increase in IL-10 expression (5.5x at A(600) 0.1). Inducible nitric oxide synthetase (iNOS) and arginase (I+II) genes were also significantly up-regulated upon exposure to all strains. However, iNOS:arginase ratio was elevated in the effector mutant challenge. These results suggest that A. salmonicida subsp. salmonicida may enhance survival within the host cell through polarization of macrophages/leucocytes to an alternative, rather than classical, activation state. Furthermore, the short-term survival and lack of T-cell signalling cytokine stimulation in DeltaascC, may help explain its inefficiency at providing protection to subsequent wild type challenge.
