The ubiquitin ligase NKLAM promotes apoptosis and suppression of cell growth

TL;DR

The protein NKLAM, an E3 ubiquitin ligase, inhibits cell growth and induces apoptosis, partly by reducing levels of the c-Myc protein.

Contribution

This study identifies a critical cysteine residue in NKLAM essential for its ubiquitin ligase activity and reveals its role in suppressing cell growth and inducing apoptosis.

Findings

NKLAM inhibits cell proliferation and reduces c-Myc levels, promoting proteasomal degradation.

The C301A mutant of NKLAM lacks ligase activity but the C301S mutant still induces apoptosis.

NKLAM induces apoptosis through annexin-V staining and caspase activation.

Abstract

Natural killer lytic-associated molecule (NKLAM), also known as RNF19b, is a member of the RING-in between-RING-RING (RBR) E3 ubiquitin ligase family and plays a pivotal role in immune regulation. We identified a critical cysteine residue at position 301 essential for NKLAM's ubiquitin ligase function. Site-directed mutagenesis of this residue to serine or alanine abrogated the ligase activity of NKLAM. Utilizing inducible expression systems in two different cell lines, HEK293 embryonic kidney cells and K562 myeloid leukemia cells, we demonstrated that wild-type (WT) NKLAM, but not the catalytically inactive NKLAM alanine mutant (C301A), inhibited cellular proliferation, as evidenced by reduced cell numbers and decreased metabolic activity. Moreover, NKLAM expression led to a significant decrease in the abundance and stability of the proto-oncogene c-Myc, a key regulator of proliferation. NKLAM facilitated the proteasomal degradation of c-Myc, with a reduction in c-Myc half-life from 27 min to 12 min and restoration of c-Myc levels upon proteasome inhibition. Notably, prolonged NKLAM expression induced apoptosis, measured by annexin-V staining and caspase activation. Strikingly, the serine mutant, C301S, while lacking ubiquitin ligase activity, induced apoptosis comparable to WT NKLAM, highlighting an alternative pathway for NKLAM-mediated inhibition of cellular homeostasis. Our findings indicate that NKLAM is a cytolytic protein with multifaceted roles in cellular proliferation and apoptosis.

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