Ras related c3 botulinum toxin substrate 1 rac1
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Ras-related C3 botulinum toxin substrate 1 (RAC1). Aksam Ahmad. RAC1 Synopsis. small G protein (GTPase) member of the Ras superfamily of guanosine triphosphate (GTP) binding proteins cell functions: regulation actin polymerization in membrane ruffling

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Rac1 synopsis
RAC1 Synopsis

  • small G protein (GTPase)

  • member of the Ras superfamily of guanosine triphosphate (GTP) binding proteins

  • cell functions:

    • regulation actin polymerization in membrane ruffling

    • formation of focal adhesion (FA) complexes

    • formation of lamellipodia

    • regulation of NADPH oxidase activity in NADPH complex


Cell movement

  • essential element of many cellular processes such as phagocytosis, wound repair, embryogenesis, immunological responses to tumor metastasis

  • critical step is extension of protrusions in the direction of the desired move, which is accomplished by membrane ruffling and the formation of lamellipodia

  • Rac1 is the primary Rho-GTPase responsible for regulating the cytoskeletal remodeling involved with lamellipodia dynamics


Structure switch i ii regions
Structure: Switch I/II Regions

  • responsible for molecular interactions

  • Switch I mainly interacts with downstream effectors, such as IQGAP1

  • also called ‘effector region’

  • Switch II mainly interacts with activating proteins (GEFs)

Switch II

Switch I

PDB ID: 1mh1


Structure insert region
Structure: Insert Region

  • only present in Rho family (residues 124-135)

  • between beta-strand 5 and alpha-helix 4

  • essential for mitogenesis and apoptosis

  • regulating downstream effector interactions, specifically NADPH oxidase

insert region

PDB: 1mh1


Rac1 compared to rab5
RAC1 compared to RAB5

  • Rab5 is also a member of the Ras superfamily of GTP binding proteins

  • Whereas Rac1 belongs to the Rho subfamily, Rab5 belongs to the Rab subfamily

  • Rab subfamily is largest subfamily of Ras GTP proteins

  • All Ras proteins have nearly identical structure, except for Rho subfamily

  • INSERT REGION only present in Rho family

insert region

Rac1: 1mh1 (red)

Rab5: 1r2q (purple)


Structure c terminus
Structure: C-Terminus

  • C-terminus participates in the binding of Rac1 to the membrane

    • necessary particularly in NADPH complex

  • members of Rho subfamily (and to lesser extent Ras superfamily) share ~92% sequence homology; sequence divergence occurs mainly in C-Termini

  • Rac1 contains polybasic amino acid residues while other Rho proteins are less basic


Rac1 activation
RAC1 Activation

  • Rac1 activation is regulated by guanosine nucleotide exchange factors (GEFs)

    • Tiam1 is one such GEF

  • Dependent on replacing bound GDP with GTP, as Rac1 is initially bound to GDP in its inactive state

  • GTP-bound Rac1 (active) is able to interact with downstream effectors

  • GTP-ase activating proteins (GAPs) facilitate GTP hydrolysis necessary for switch from inactive to active Rac1

  • RhoGDI regulates inactive/active cycles by isolating GDP-Rac1








Rac1 interaction tiam1 gef
RAC1 Interaction TIAM1 (GEF)

PDB ID: 1foe

Rac1 (red)

Switch Regions (green)

Tiam1 (blue)

DH Domain


Cell adhesion and rac1
Cell Adhesion [and RAC1]






beta- catenin

Actin Filaments

beta- catenin


alpha- catenin

beta- catenin


Cell adhesion cont

ACTIN FILAMENTS linked to membrane through VINCULIN


E-CADHERIN essentially plays biggest role in ‘gluing’ cells together



Rac1 in nadph complex
RAC1 in NADPH Complex

  • Consists of p40phox, p47phox, p67phox, b558 (membrane-bound heterodimer composed of gp91phox and p22phox) and Rac1

  • Activation of complex occurs when cell is exposed to inflammatory stimuli and phagocytes are activated

  • p47phox & p67phox are phosphorylated (SO4); Rac1 is activated (becomes GTP bound)

  • These three components then translocate to membrane and form complex with b558

  • Complex then initiates electron flow and superoxide production


Nadph complex
NADPH Complex

  • Note:

  • p67phox and p47phox are phosphorylated

  • Rac1 is in its active form bound to GTP

  • Rac1 is bound to membrane via C-Terminus

  • b558 is composed of p22phox and gp91phox


  • superoxide is generated









NADPH + 2O2 ↔ NADP+ + 2O2°- + H+


Superoxide significance
Superoxide Significance

  • facilitates destruction of invading microorganisms during phagocytosis

  • capable of killing bacteria and fungi due to its ability to react with other compounds

  • Flipside:

    • can lead to increased production of reactive oxygen species (ex: hydrogen peroxide), which cause protein oxidation, DNA damage, and neuronal cell death



  • novel splice variant of Rac1

  • different from mutation; alternative sequences that can occur naturally

  • contains extra 19 amino acid insertion (between codons 75 and 76 and end of Switch II region)

  • associated with tumors and human cancers..

Switch I Regions

Rac1: 1mh1 (purple)

Rac1b: 1ryf (yellow)


Rac1b structure comparison
RAC1B Structure Comparison


Rac1b:GDP (purple) Rac1b:GppNHp (red) Rac1:GppNHp (brown)


Rac1b properties
RAC1B Properties

  • expressed in human breast and colon carcinoma cells

  • impaired GTPase activity and interaction with downstream effectors; no interaction with Rho GDI

  • causes increase in cellular reactive oxygen species (ROS)  stimulate expression of transcription factor Snail and EMT  cause oxidative damage to DNA and genomic instability leading to cancer

  • promotes growth of NIH3T3 cells (cells that maintain growth of extracellular matrix of tissues)

  • not involved in all important lamellipodia formation, which therefore impairs cell mobility


Rac1b as a biomarker
RAC1B as a Biomarker

  • Rac1b has been identified as a promising early stage biomarker for matrix metalloproteinase (MMP)-induced malignancies

    • such malignancies include breast, colon, prostate, kidney, skin, stomach, ovary, and lung cancers

    • biomarker is any substance that serves as an indicator of biologic state

  • Rac1b is NOT generated in non-malignant cells

  • unlike most uncogenic isforms induced in cancers, Rac1b becomes more highly activated



  • Rac1b associated malignancies are highly therapeutic

  • Labs (including Bissell Lab at Berkeley) have been able to develop Rac1b antibody

  • Rac1b siRNA developed as well

    • involved in the RNA interference (RNAi) pathway and more specifically interferes with expression of specific gene



  • (1) "Actin Cytoskeleton". April 20, 2008 <http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/ part1/actin.htm>.

  • (2) Fiegen, Dennis. "Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase". April 20, 2008 <http://www.jbc.org/cgi/content/full/279/6/4743>.

  • (3) Freeman, Jennifer. "Rac Insert Region Is a Novel Effector Region That Is Implicated in the Activation of NADPH Oxidase". April 20, 2008 <http://www.jbc.org/cgi/content/abstract/271/33/19794>.

  • (4) "RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1". April 20, 2008 <http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602048>.

  • (5) Williams, Emily. "Human Rac1 in Complex with p67phox". April 20, 2008 <http://biology.kenyon.edu/BMB/Chime2/2003/RAC1/FRAMES/index.htm>.

  • (6) Worthylake, David. "Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1". April 20, 2008 <http://www.nature.com/nature/journal/v408/n6813/full/408682a0.html>.