E3 ubiquitin ligase NKLAM is a macrophage phagosome protein and plays a role in bacterial killing

Abstract

Macrophages are a critically important component of the innate and adaptive immune systems. They are equipped with oxidative and non-oxidative mechanisms to kill ingested pathogens. Natural Killer Lytic-Associated Molecule (NKLAM) is an E3 ubiquitin ligase expressed in macrophages and natural killer cells. We show that NKLAM expression in macrophages was enhanced by Toll-like receptor agonists and pro-inflammatory cytokines. Using confocal microscopy, we found that NKLAM colocalized with ingested E. coli. In assays using IgG-opsonized latex beads as targets, we demonstrated that NKLAM translocated to the phagosome early during maturation at a time that coincided with elevated levels of ubiquitinated phagosome proteins. In killing assays with bone marrow-derived macrophages from wild type and NKLAM-deficient mice, we found that NKLAM-deficient macrophages demonstrated less killing of Escherichia coli than wild type macrophages. Collectively, our data show that NKLAM is a novel component of macrophage phagosomes and is involved in macrophage bactericidal functions.

E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression

“…Our previous studies demonstrated that NKLAM-KO macrophages are inherently defective in killing E. coli (Lawrence and Kornbluth, 2012). Macrophages employ a number of bactericidal mechanisms to combat pathogens; these include phagosome acid hydrolases, phagosomal pH reduction and NO production (Flannagan, et al, 2009).…”

“…Recent studies from our laboratory demonstrated that the RBR family member NKLAM plays an important role in regulating macrophage killing of E. coli (Lawrence and Kornbluth, 2012). However, precisely how NKLAM is involved in the murine macrophage killing response has not been elucidated.…”

“…NKLAM is weakly expressed under resting conditions but is upregulated by cytokines (e.g. IFNγ) and bacterial products such as LPS (Lawrence and Kornbluth, 2012). NK cells from NKLAM-deficient (KO) mice have diminished anti-tumor cytolytic activity (Hoover, et al, 2009).…”

“…We found that macrophages (bone marrow-derived and peritoneal) from NKLAM-KO mice are significantly defective in killing Escherichia coli . Despite its localization to phagosomes, NKLAM does not regulate bacteria uptake or phagosome acidification (Lawrence and Kornbluth, 2012). Thus, NKLAM is likely involved in regulating some other macrophage killing mechanism within the phagosome.…”

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

“…The C-terminal portion of NKLAM is devoid of any known domains. Studies from our laboratory have shown that NKLAM colocalizes with NK cell granules and bacteria-containing phagosomes in macrophages [1,4]. NKLAM is weakly expressed in unstimulated cells and upregulated by cytokines (e.g.…”