Insights into K-Ras 4B regulation by post-translational lysine acetylation

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Ras proteins function as molecular switches, cycling between active (GTP-bound) and inactive (GDP-bound) states.
• Ras mutations, particularly affecting the 'off-switch', are prevalent in human cancers.
• Lysine acetylation at K104 of K-Ras 4B was recently identified, with a hypothesis that it impairs SOS-catalyzed guanine-nucleotide exchange, potentially reducing oncogenicity.

Purpose of the Study:

• To synthesize site-specifically acetylated K-Ras 4B (wildtype and G12V mutant) to experimentally test the impact of K104 acetylation on guanine-nucleotide exchange.
• To investigate the role of other acetylation sites, such as K147, in regulating K-Ras 4B function.
• To determine if K-Ras 4B, acetylated at K104 or K147, is a substrate for deacetylases Sirt2 and HDAC6 in vitro.

Main Methods:

• Utilized the genetic-code expansion technique to prepare site-specifically K104-acetylated K-Ras 4B wildtype and G12V mutant proteins.
• Performed in vitro assays to measure SOS-catalyzed guanine-nucleotide exchange on wildtype and mutant K-Ras 4B.
• Employed mass spectrometry to identify additional acetylation sites on K-Ras 4B.
• Conducted in vitro deacetylation assays using Sirt2 and HDAC6.

Main Results:

• K104 acetylation did not affect SOS-catalyzed nucleotide exchange for either wildtype or G12V K-Ras 4B, indicating glutamine is a poor mimetic for acetylation at this site.
• Lysine acetyltransferases CBP and p300 successfully acetylated both wildtype and G12V K-Ras 4B in vitro.
• Identified K147 as another acetylation site in K-Ras 4B, but its acetylation also did not impact intrinsic or SOS-catalyzed nucleotide exchange.
• Neither Sirt2 nor HDAC6 deacetylated K-Ras 4B acetylated at K104 or K147 in vitro.

Conclusions:

• K104 acetylation does not inhibit K-Ras 4B's interaction with SOS or its guanine-nucleotide exchange activity.
• The previously hypothesized mechanism of K104-acetylation impairing oncogenicity via SOS inhibition is not supported by these findings.
• K-Ras 4B acetylation at K104 and K147 does not appear to be regulated by Sirt2 and HDAC6 in vitro, suggesting alternative regulatory mechanisms or enzymes may be involved.