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
– formation of focal adhesion (FA) complexes
– formation of lamellipodia
– regulation of NADPH oxidase activity in NADPH
complex
(4)
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
(1)
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)
(4)
Switch II
Switch I
PDB ID:
1mh1
Structure: Insert Region
insert
region
PDB: 1mh1
(3)
• 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
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
insert
region
Rac1: 1mh1 (red)
Rab5: 1r2q (purple)
(3)
- All Ras proteins have nearly
identical structure, except for Rho
subfamily
- INSERT REGION only present in
Rho family
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)
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
inactive
(6)
active
RAC1 Interaction TIAM1 (GEF)
PDB ID: 1foe
Rac1 (red)
Switch
Regions
(green)
Tiam1 (blue)
DH Domain
(6)
Cell Adhesion [and RAC1]
PLASMA MEMBRANE
E-cadherin
Tiam1
betacateni
n
Actin Filaments
IQGAP1
RAC1
betacateni
n
RAC1
alphacateni
n
betacateni
n
CYTOSOL
CELL ADHESION [cont.]
-ACTIN FILAMENTS linked to
membrane through VINCULIN
-VINCULIN links to ECADHERIN with the
ALPHA/BETA-CATENINS
-E-CADHERIN essentially plays
biggest role in ‘gluing’ cells
together
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/actin.htm
(1)
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)
NADPH Complex
Note:
p47phox
p67phox
b558
membrane
Rac1
p40phox
SO4
NADPH + 2O2 ↔ NADP+ + 2O2°- +
H+
(5)
GTP
- p67phox and p47phox
are phosphorylated
- Rac1 is in its active form
bound to GTP
- Rac1 is bound to
membrane via CTerminus
- b558 is composed of
p22phox and gp91phox
- SIGNIFICANT RESULT:
superoxide is generated
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)
RAC1B
Switch I
Regions
Rac1: 1mh1 (purple)
Rac1b: 1ryf (yellow)
(2)
• 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..
RAC1B Structure Comparison
http://www.jbc.org/cgi/content/full/279/6/4743
Rac1b:GDP (purple) Rac1b:GppNHp (red) Rac1:GppNHp (brown)
(2)
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)
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)
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)
REFERENCES
•
•
•
•
•
•
(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>.