Natural Killer Lytic-Associated Molecule (NKLAM): An E3 Ubiquitin Ligase With an Integral Role in Innate Immunity

Abstract

Natural Killer Lytic-Associated Molecule (NKLAM), also designated RNF19B, is a unique member of a small family of E3 ubiquitin ligases. This 14-member group of ligases has a characteristic cysteine-rich RING-IBR-RING (RBR) domain that mediates the ubiquitination of multiple substrates. The consequence of substrate ubiquitination varies, depending on the type of ubiquitin linkages formed. The most widely studied effect of ubiquitination of proteins is proteasome-mediated substrate degradation; however, ubiquitination can also alter protein localization and function. Since its discovery in 1999, much has been deciphered about the role of NKLAM in innate immune responses. We have discerned that NKLAM has an integral function in both natural killer (NK) cells and macrophages in vitro and in vivo. NKLAM expression is required for each of these cell types to mediate maximal killing activity and cytokine production. However, much remains to be determined. In this review, we summarize what has been learned about NKLAM expression, structure and function, and discuss new directions for investigation. We hope that this will stimulate interest in further exploration of NKLAM.

Keywords: NKLAM; RNF19B; cytotoxicity; innate immunity; macrophage; natural killer; phagocytosis; ubiquitin ligase.

FIGURE 1

Diagram of human NKLAM (RNF19B) gene organization. NKLAM is encoded by 9 exons (depicted as blue bars) spanning 28 kb in the reverse strand of chromosome 1. Exons 1–3 encode intrinsically disordered region 1 (IDR1) and the three RINGS of the ligase domain. Exons 4 and 5 encode the two transmembrane domains (gray bars), while IDR 2 is encoded across the remaining exons 6–9. Mouse NKLAM, located on chromosome 4, has the same genomic structure. Human NKLAM mRNA isoform 1 encodes a protein with a predicted molecular weight of 77.9 kDa. Alternative splicing of exon 9 results in a shorter mRNA isoform 2 encoding a protein with a predicted molecular weight of 62.7 kDa. Mice only express the long form of NKLAM protein.

FIGURE 2

Structure/function of NKLAM protein. The RING1-IBR-RING2 (RBR) domain has the ubiquitin ligase activity. RING2 contains the highly conserved, active site catalytic cysteine (C302), which is the defining feature of this family of RBR ligases. In addition to the two transmembrane (TM) domains, the N- and C-terminal ends of NKLAM are intrinsically disordered regions (IDR). Bioinformatic analysis using the DEPICTER server indicate the possible roles of the IDR in protein, DNA, and RNA binding.

FIGURE 3

Proposed model of NKLAM function in NK cells. The cytolytic granules of resting NK cells contain little to no NKLAM. Upon cytokine exposure or target stimulation, activated NK cells rapidly transcribe and translate NKLAM, which is embedded in cytolytic granule membranes. Smaller, intragranular vesicles also have NKLAM in their membranes. NK-tumor interaction releases granule contents (perforin, granzymes) into the tumor cell at the immunological synapse. Intragranular vesicles are also released; these now called extracellular vesicles (EVs) contain NKLAM, as well as perforin and granzymes, and enter the tumor cell by endocytosis. Granzymes induce caspase activation; NKLAM ubiquitinates and degrades the tumor survival protein UCKL-1. This combination of perforin, granzymes, and NKLAM ensures death of the tumor cell. Red circles: EVs; gray bars: NKLAM; green squares, blue ovals: cytolytic proteins granzymes A and B; yellow bars: perforin; purple bar: UCKL-1; yellow circles: ubiquitin.

FIGURE 4

NKLAM in macrophage immune function. Upon exposure to IFNγ, STAT1 is phosphorylated and NKLAM is transiently associated with the IFNγ-IFNGR complex at the plasma membrane. In this location, NKLAM is in close proximity to STAT1, which would allow NKLAM to ubiquitinate STAT1 or other proteins to promote STAT1 transcriptional activity. After ingestion of bacterial targets, NKLAM is localized to the phagosome membrane with the catalytic RING2 domain and C-terminal tail facing the cytoplasm. This would provide NKLAM with access to phagosome membrane proteins and cytosolic target proteins, as well as bacteria that escape the phagosome. Red circles; phosphotyrosine, yellow circles; ubiquitin. Immunomicrograph insert: Macrophage phagosomes after ingestion of fluorescent-labeled E. coli (red). NKLAM staining: green; Image J-defined co-localization of E. coli and NKLAM: white. Adapted from Lawrence, D.W., and Kornbluth, J. E3 ubiquitin ligase NKLAM is a macrophage phagosome protein and plays a role in bacterial killing. Copyright 2012, with permission from Elsevier and Lawrence D.W., and Kornbluth, J. E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity. Copyright 2016, with permission from Elsevier.