The death-inducing activity of RIPK1 is regulated by the pH environment

Area of Science:

• Cellular biology
• Molecular signaling
• Inflammation research

Background:

• Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of cell survival and death pathways, particularly in response to tumor necrosis factor (TNF).
• RIPK1 activity is modulated by post-translational modifications like ubiquitination and phosphorylation.
• The influence of environmental factors on RIPK1-mediated cell death remains incompletely understood.

Purpose of the Study:

• To investigate the impact of extracellular and intracellular pH on RIPK1 kinase activity and TNF-induced cell death.
• To elucidate the specific mechanisms by which pH influences RIPK1-dependent signaling pathways.
• To identify potential pH-sensitive residues within RIPK1 involved in regulating its kinase activity.

Main Methods:

• Utilized cell culture models exposed to varying pH conditions.
• Assessed TNF-induced cell death, including necroptosis and apoptosis, via biochemical assays.
• Analyzed RIPK1 kinase activation, autophosphorylation at Ser166, complex formation (Complex I and Complex II), ubiquitination, and NF-κB activation.
• Employed site-directed mutagenesis to probe the role of specific histidine residues in pH sensitivity.

Main Results:

• Acidic extracellular pH, leading to decreased intracellular pH, significantly impaired RIPK1 kinase activation and autophosphorylation at Ser166.
• This pH-dependent inhibition reduced the formation of the cytosolic death-inducing complex II, thereby suppressing RIPK1-dependent necroptosis and apoptosis.
• Low pH did not affect the formation of the membrane-bound TNFR1-containing signaling complex I, RIPK1 ubiquitination, or NF-κB activation.
• Abolition of pH sensitivity was observed in cells expressing a RIPK1 mutant with a modified His151 residue, indicating its role as a proton sensor.

Conclusions:

• Extracellular and intracellular pH represent critical environmental factors that can tune the kinase activity of RIPK1.
• Acidic pH inhibits RIPK1-dependent cell death pathways by impairing kinase activation and complex II formation, while sparing Complex I and NF-κB signaling.
• His151 in RIPK1 acts as a crucial proton acceptor, mediating the pH-dependent regulation of RIPK1 activity and subsequent cell fate decisions.