A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction

Area of Science:

• Molecular biology
• Cell signaling
• Redox biology

Background:

• Nitric oxide (NO) is a critical signaling molecule involved in numerous physiological processes.
• p21(ras) is a key regulator of signal transduction pathways.
• The interaction between NO and p21(ras) and its role in signaling initiation are not fully understood.

Purpose of the Study:

• To identify the specific molecular site of interaction between nitric oxide (NO) and p21(ras).
• To elucidate the role of this interaction in initiating signal transduction.
• To investigate the functional consequences of NO-mediated modification of p21(ras).

Main Methods:

• Site-directed mutagenesis to create p21(ras)C118S mutant.
• S-nitrosylation assays to detect modification of p21(ras).
• Guanine nucleotide exchange assays to measure p21(ras) activity.
• Mitogen-activated protein kinase (MAPK) activity assays in transfected cells.

Main Results:

• p21(ras) undergoes S-nitrosylation specifically at Cys118.
• A Cys118 to serine mutant (p21(ras)C118S) abrogated S-nitrosylation.
• NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras) but not on p21(ras)C118S.
• NO-related species failed to stimulate MAPK activity in cells expressing p21(ras)C118S.

Conclusions:

• Cysteine 118 is a critical site for redox regulation of p21(ras) by nitric oxide.
• S-nitrosylation of Cys118 by NO triggers guanine nucleotide exchange and downstream signaling.
• This finding reveals a novel mechanism for NO-mediated regulation of cell signaling.