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Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

G Aaron Hobbs¹, Harsha P Gunawardena¹, Rachael Baker¹

¹Department of Biochemistry and Biophysics; University of North Carolina; Chapel Hill, NC USA.

|September 14, 2013

Summary

This summary is machine-generated.

Monoubiquitination (mUb) of KRas at position 147 activates it by impairing GTPase activating protein (GAP) function. Linker properties, not just modification, determine GAP activity, requiring a minimum length of 7-8 residues.

Keywords:

GTP hydrolysis GTPase Activating Proteins Post-translational modifications glutathiolation monoubiquitination

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Area of Science:

Molecular Biology
Cell Signaling
Protein Biochemistry

Background:

KRas activation is crucial in cell signaling.
Monoubiquitination (mUb) of KRas at position 147 has been identified as an activating modification.
This mUb impairs the function of GTPase activating protein (GAP), leading to Ras dysregulation.

Purpose of the Study:

To elucidate the mechanism by which mUb impairs GAP-mediated Ras downregulation.
To investigate the role of modifications at position 147 of KRas on Ras sensitivity to GAP function.
To determine the structural requirements of the linker region in mUb-mediated Ras activation.

Main Methods:

Site-specific modifications at position 147 of KRas.
Assays to measure Ras sensitivity to GAP-mediated GTP hydrolysis.
Comparative analysis of different linker lengths and properties (ubiquitin, PDZ2 domain).

Main Results:

Small chemical modifications at position 147 did not affect GAP-mediated hydrolysis.
Ligation of KRas to ubiquitin or PDZ2 with native linkers resulted in defective GAP-mediated GTP hydrolysis.
Restoration of GAP activity was observed when using a shorter, stiffer linker (PDZ2 domain).
A minimum linker length of 7 to 8 residues is required for the GAP defect.

Conclusions:

The properties of the linker region connecting the modifying moiety to KRas are critical for GAP function.
Modification-induced Ras activation is dependent on linker length and flexibility.
These findings provide mechanistic insights into KRas regulation by mUb and potential therapeutic targets.