1. Ras-related C3 botulinum toxin substrate 1 (RAC1)
Aksam Ahmad

2. 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
Rho family more specifically
(4)

3. 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
(1)

4. 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
Switch I = Effector Region (cause of main interactions with effectors)
PDB ID: 1mh1
(4)

5. Structure: Insert Region
only present in Rho family (residues )
between beta-strand 5 and alpha-helix 4
essential for mitogenesis and apoptosis
regulating downstream effector interactions, specifically NADPH oxidase
Has no counterpart in RAS!
insert region
PDB: 1mh1
(3)

6. 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)
(3)

7. 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
(4)

8. 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
RAC1
GDP
GTP
active
inactive
(6)

9. RAC1 Interaction TIAM1 (GEF)
PDB ID: 1foe
Rac1 (red)
Switch Regions (green)
Tiam1 (blue)
DH Domain
(6)

10. Cell Adhesion [and RAC1]
PLASMA MEMBRANE
E-cadherin
Tiam1
IQGAP1
RAC1
beta- catenin
Actin Filaments
beta- catenin
IQGAP1 is an effector. Tiam1 is an GEF (guanine nucleotide exchange factor). Catenins help to bind actin (necessary for cell adhesion). Actin are involved in cadherin based adhesion, shown in next slide.
RAC1
alpha- catenin
beta- catenin
CYTOSOL

11. CELL ADHESION [cont.]
ACTIN FILAMENTS linked to membrane through VINCULIN
VINCULIN links to E-CADHERIN with the ALPHA/BETA-CATENINS
E-CADHERIN essentially plays biggest role in ‘gluing’ cells together
Vinculin is a protein.
(1)

12. 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
(5)

13. 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
SIGNIFICANT RESULT:
superoxide is generated
p47phox
p67phox
b558
membrane
Rac1
p40phox
SO4
GTP
NADPH + 2O2 ↔ NADP+ + 2O2°- + H+
(5)

14. 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
(5)

15. RAC1B 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)
(2)

16. RAC1B Structure Comparison
switch II region and the adjacent 19-amino acid insertion are not resolved and therefore not included. MAIN difference is displacement of switch 1 region from nucleotide binding site.
This affects its ability in binding interactions and explains the dramatic difference in properties found in Rac1b.
Rac1b:GDP (purple) Rac1b:GppNHp (red) Rac1:GppNHp (brown)
(2)

17. 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
(2)

18. 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
(2)

19. Treatment 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
(2)

20. REFERENCES
(1) "Actin Cytoskeleton". April 20, 2008 < part1/actin.htm>.
(2) Fiegen, Dennis. "Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase". April 20, 2008 <
(3) Freeman, Jennifer. "Rac Insert Region Is a Novel Effector Region That Is Implicated in the Activation of NADPH Oxidase". April 20, 2008 <
(4) "RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1". April 20, 2008 <
(5) Williams, Emily. "Human Rac1 in Complex with p67phox". April 20, 2008 <
(6) Worthylake, David. "Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1". April 20, 2008 <