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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:
The Ras Gene02:38

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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.
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Cell polarity is the asymmetric distribution of cellular and membrane components, making one side of the cell different from the other. This polarity is essential to many processes such as embryogenesis, axon migration, glucose transport across epithelial cells, and directional cell migration. A migrating cell responds to intracellular or extracellular signals via molecular cascades that reorganize the actin cytoskeleton to establish this polarity. In these cells, the Rho family proteins Cdc42,...
Rab Proteins01:14

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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.
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PI3K/mTOR/AKT Signaling Pathway01:22

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The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...

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Related Experiment Video

Updated: Jun 4, 2026

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
07:49

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

Published on: July 17, 2019

Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1.

Catherine L Hutchinson1, Peter N Lowe, Stephen H McLaughlin

  • 1Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.

Biochemistry
|March 1, 2011
PubMed
Summary

Protein kinase C-related kinases (PRKs) bind Rho GTPases via HR1 domains. Mutagenesis revealed only one HR1a contact site is crucial for RhoA binding, with HR1b not contributing.

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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

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Last Updated: Jun 4, 2026

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07:49

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
11:28

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Published on: March 31, 2012

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • Protein kinase C-related kinases (PRKs) are serine/threonine kinases.
  • PRKs bind Rho family small G proteins through HR1 domains.
  • PRK1 possesses three tandem HR1 domains (HR1a, HR1b, HR1c).

Purpose of the Study:

  • To investigate the functional relevance of two potential contact sites in the HR1a domain for RhoA binding.
  • To determine if the HR1b domain contributes to RhoA binding.
  • To assess the biophysical properties, including thermal stability, of HR1a and HR1b domains.

Main Methods:

  • Alanine scanning mutagenesis of PRK1 HR1 domains.
  • In-solution binding assays to assess RhoA interaction.
  • Differential scanning fluorimetry to measure thermal stability.

Main Results:

  • Mutagenesis identified a single critical contact site within the HR1a domain for RhoA interaction.
  • The HR1b domain was found to have no significant role in RhoA binding.
  • HR1b exhibited greater thermal stability compared to HR1a.

Conclusions:

  • Only one of the two previously proposed contact sites in HR1a is essential for RhoA binding in solution.
  • The HR1b domain does not participate in RhoA binding.
  • Differential biophysical properties, particularly thermal stability, may dictate the distinct roles of HR1 domains in G protein interaction.