Related Experiment Video

Updated: May 7, 2026

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
10:27

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Published on: March 9, 2012

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.

Small Gtpases
|September 14, 2013
PubMed

Insights

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.

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.
Keywords:
GTP hydrolysisGTPase Activating ProteinsPost-translational modificationsglutathiolationmonoubiquitination

More Related Videos

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta10:27

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

Related Concept Videos

The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Rab Proteins01:14

Rab Proteins

Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
GTPases and their Regulation02:14

GTPases and their Regulation

Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...