E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity

Abstract

Signal transducer and activator of transcription 1 (STAT1) is critically important for the transcription of a large number of immunologically relevant genes. In macrophages, interferon gamma (IFNγ) signal transduction occurs via the JAK/STAT pathway and ends with the transcription of a number of genes necessary for a successful host immune response. The predominant mechanism of regulation of STAT1 is phosphorylation; however, there is a growing body of evidence that demonstrates STAT1 is also regulated by ubiquitination. In this report we show that JAK1 and STAT1 in macrophages deficient in an E3 ubiquitin ligase termed Natural Killer Lytic-Associated Molecule (NKLAM) are hyperphosphorylated following IFNγ stimulation. We found NKLAM was transiently localized to the IFNγ receptor complex during stimulation with IFNγ, where it bound to and mediated K63-linked ubiquitination of STAT1. In vitro nucleofection studies demonstrated that STAT1-mediated transcription was significantly reduced in NKLAM-KO macrophages. There was no obvious defect in STAT1 nuclear translocation; however, STAT1 from NKLAM-KO macrophages had a reduced ability to bind a functional gamma activation DNA sequence. There was also less mRNA expression of STAT1-mediated genes in NKLAM-KO macrophages treated with IFNγ. Our results demonstrate for the first time that NKLAM is a positive regulator of STAT1-mediated transcriptional activity and is an important component of the innate immune response.

Reduced inflammation and cytokine production in NKLAM deficient mice during Streptococcus pneumoniae infection

“…More recent research from our laboratory demonstrated that NKLAM associates with STAT1 and mediates its K63-linked ubiquitination [ 8 ]. We have also shown that NKLAM plays significant role in facilitating the binding of STAT1 to consensus binding sites (GAS; gamma activated sites) within the promoter region of iNOS.…”

“…We have also shown that NKLAM plays significant role in facilitating the binding of STAT1 to consensus binding sites (GAS; gamma activated sites) within the promoter region of iNOS. Without NKLAM, IFNγ-stimulated NKLAM-KO macrophages express significantly less iNOS than wild type (WT) macrophages [ 8 ]. In response to bacteria or bacterial products, STAT1 is phosphorylated on tyrosine 701, dimerizes and translocates to the nucleus where it is involved in the transcription of many immunologically important genes.…”

“…There is an emerging body of evidence suggesting that RBR ubiquitin ligases are involved in regulating the expression of pro-inflammatory cytokines. Studies from our laboratory have shown that NKLAM is involved in enhancing the expression of IFNγ or RANTES/CCL5 in response to LPS and IFNγ, respectively [ 8 , 12 ]. The knockdown of E3 ubiquitin ligase Parkin with siRNA or the use of Parkin-KO mice demonstrated that Parkin, in part, regulates the expression of lung IL-6 and TNFα as well as the expression of IL-6 and IL-8 from endothelial cells [ 13 ].…”

Degradation of CFTR-F508del By the Ubiquitin E2 Conjugating Enzyme UBE 2L6 and the E3 Ligase RNF19B

“…RNF19B is a Ring finger protein family of E3 ligases, which interacts with E2 conjugating enzyme(s) and substrates, facilitating the transfer of an activated ubiquitin to the substrate [29,30]. RING finger E3 ligases have a RING structure that is involved in ubiquitinating its substrate.…”

The BLIMP1 – EZH2 nexus in a non-Hodgkin lymphoma

“…When we plotted signal for 315 H3K27me3 at ± 3 kb flanking BLIMP1 peaks, only a small level of enrichment was 316 observed ( Fig 4B). However, when we assigned the peaks from the respective 317 experiments to genes, a significant overlap of 261 genes emerged between BLIMP1 318 and H3K27me3 in RP cells ( Fig 4C), including B-cell genes PIK3CD and POU2F2 319 (OCT2), the STAT1 activator RNF19B (NKLAM) (Lawrence & Kornbluth, 2016), and 320 the cell-growth inhibitor NUB1 ( Fig 4D). 321…”