Dr. Van-Lamarche (Rho GTPases) lect21-24 Flashcards
1
Q
name the 4 main cellular responses to a signal
A
survive, divide, differentiate, die
2
Q
what kind of ligands interact with cell surface vs intracellular receptors?
A
hydrophilic ligands with cell-surface; hydrophobic ligands with intracellular
3
Q
name and briefly describe the 4 types of extracellular signalling
A
- contact dependent: cells touch. ex eph/ephrins
- paracrine: secreted ligands over short distance
- synaptic: long distance signalling with neurotransmitters
- endocrine: long distance signalling via hormones travelling in blood
4
Q
name the 3 classes of cell-surface receptors
A
- ion-channel-linked
- GPCRs
- enzyme-linked receptors
5
Q
what process are ion-channel linked receptors involved in?
A
synaptic connections: they change ion permeability of the PM changing escitability of the postsynaptic cell
6
Q
how many TM domain do enzyme-linked receptors have?
A
1 short TM domain
7
Q
how do enzyme-linked receptors work?
A
2 options:
1. ligand binds in form of a dimer to a dimerized receptor: receptor has catalytic activities itself
2. single signal molecule binds: receptor activates an enzyme which has catalytic acitivty
8
Q
what are relay proteins? give examples
A
enzymes, kinases/phosphatases, mediates signaling via phosphor/dephosphorylation
9
Q
what are scaffold proteins?
A
no enzymatic activities, multiple signaling domains to mediate interactions, induce large cluster of protein complexes
10
Q
what are amplifiers?
A
GTPases, Kinases, phosphatases: enzymes on/off, small GTPases, interact with intracellular mediators that have their downstream effect
11
Q
what do intracellular mediators can amplifiers interact with?
A
integrators! (substrates or effectors of amplifiers)
12
Q
kinases required what to act?
A
ATP
13
Q
how can a signaling complex get assembled? (2 ways)
A
- via scaffolding protein
- via the activated receptor itself that binds multiple proteins
14
Q
name the most common amplifier
A
GTPase!
15
Q
name 2 differences between kinases/phosphatases and GTPases
A
- Kinases use ATP, GTPases use GTP
- the phosphate released does not go to phosphorylate another protein with a kinase
16
Q
what is the role of integrators?
A
modulate the interaction between signaling pathways
17
Q
how many families of RTKs are there?
A
7
18
Q
what are characteristics of RTKs structure?
A
- long intracellular domain containing the tyrosine kinase domain
- short TM domain
19
Q
most RTKs are what kind of receptors?
A
growth factor and hormone receptors
20
Q
what are the major cellular roles in which growth factor and hormone receptors are involved in?
A
cell proliferation, cell survival, cell motility, cell differentiation
21
Q
dysregulation of RTKs are involved in what type of diseases?
A
cancer
22
Q
the addition of a negative charge (phosphate) to a protein can lead to what?
A
change in conformation, stimulation/inhibition of proteins, protein interactions
23
Q
what aa get phoosphorylated?
A
tyrosine, serine, threonine
24
Q
briefly describe the general mechanism of activation of RTKs
A
- ligand binds
- oligomerization of the receptor
- trans-phosphorylation
- tyrosine kinase activation
- binding of substrates
- activation of intracellular signaling proteins
25
26
what type of proteins can RTKs bind to induce oligomerization?
proteoglycans
27
what type of proteins can RTKs bind on a different cell?
ephrins
28
how is the FULL kinase activity of RTKs unlocked?
phosphorylation of a critical tyrosine residue
29
the phosphprylation of tyrosine residues on the RTKs triggers what?
high affinity binding of proteins to the phosphorylated tyrosines
30
what kind of domain do phosphotyrosine of RTKs interact with?
SH2 domain (Src homology domain)
31
via what does the phosphotyrosine of RTKs interact with the SH2 domain?
via a small amino acid side chain
32
name some domain found in protein signaling modules
SH2, PTB, PDZ, SH3, WW, 14-3-3
33
what are protein signaling modules used for?
to build up complex networks of interacting proteins;
involved in signaling from cell surface to nucleus, protein trafficking, subcellular localisation, cell shape, cell motility, cell interactions
34
what does SH2 bind?
specific phosphotyrosine residues
35
more specifically describe how SH2 interacts with RTK
SH2 has a + charged conserved binding pocket that interacts with pY;
and a + charged specificity binding pocket that interacts with the side chain
36
what dpes 14-3-3 interact with? where does it go after?
it interacts with phosphoserine/ phosphothreonine and goes to the nucleus to induce transcription
37
what does the PH domain bind to? why?
binds PIP2 to localize protein at the plasma membrane
38
what is the scaffolding protein for the insulin receptor B subunit?
IRS-1 (insulin receptor substrate)
39
name the 2 methods mentionned for detection of protein-protein interactions via specific signaling modules
- ip
- GST pulldown
40
explain a GST pulldown assay
1. use recombinant DNA to fuse protein with GST
2. incubate your sample with GST-tagged protein
3. immobilize on glutathione sepharose beads
4. wash
5. SDS-PAGE
41
explain how immunoprecipitation works
1. tag your protein & introduce it in host cell
2. whole cell extract
3. incubate with antibody againt the tag
4. incubate with a-sephrose beads
5. centrifuge to collect beads. discard supernatant
6. wash pellet
7. separate protein by SDS-PAGE
42
what are the 5 subfamilies of the Ras small GTPases superfamily? name their function
- Ras: cell prolifration & diffferentiation
- Rho: cell morphology and movement
- Ran: nuclear import
- Rab / Arf: vesicular trafficking & secretion
43
name the prenyltransferases that can modify Ras-related proteins and what domain they each modify in what GTPases
1) farnesyltransferase: modifies CAAX domain of Ras proteins
2) type 1 geranylgeranyltransferase: CAAX domain of Rho annd Rap proteins
3) type 2 geranylgeranyltransferase: CXC or CC in Rab proteins
44
what is X in the CAAX domain of Ras vs Rho/Rap proteins that is modified by a prenyltransferase?
Ras: X = Met or Ser
Rho/Rap: x = Leu
45
how long is the farnesyl group added to Ras protein by farnesyltransferase?
15 carbons
46
via what bond is farnesyl bound to Ras proteins?
thioether/covalent bond between the carbon and sulfate at the terminal cysteine of Ras protein
47
what would be a target to inhibit Ras activity?
prenyltransferase!
48
what has a higher concentration in the cell, GTP of GDP?
GTP has a concentration that is 10x higher
49
name an important GEF for Ras
SOS protein
50
what is the structure of a Ras protein composed of?
- 6 beta sheets, 5 alpha helices
- switch helix (effector binding region)
51
what happens to the switch helix of Ras proteins when it is GTP vs GDP bound?
GTP-bound = switch helix is exposed
GFP-bound = hidden
52
Ras mutations are found in 30% of what diseases?
cancers
53
what mutation keeps Ras GTPase activated (inhibits GAP)
G12V-Ras
54
what mutation keeps Ras GTPase inhibited (inhibits GEF)?
S17N-Ras
55
you can say that Ras is what type of gene?
oncogene
56
name a GAP for Ras
neurofibronin
57
what proteins are involved in Ras activation by RTK?
1. RTK binds Shc via Yp-SH2 interaction
2. Shc binds GRB2 via Yp-SH2 interaction
3. GRB2 binds SOS via SH3-PXXP interaction
4. SOS (GEF) activates Ras
58
appart form the GEF domain and proline rich motif, what other domain does SOS have?
PH domain to bind lipids
59
what is the MAPK pathway require for?
cell division and proliferation
60
what are the main players of the MAPK pathways (name them)
- MAPKKK (Raf)
- MAPKK (Mec)
- MAPK (ERK)
61
MAPKs are what type of kinases?
serine threonine kinases
62
what are the outputs of the MAPK pathway?
MAPK phosphorylates protein to regulate their activity and/or goes to nucleus and affects gene expression
63
name ras effectors involved in cell growth and remodeling of the actin cytoskeleton
- Raf (MAPKKK)
- PI3K
- RAL-GDS
64
what are PI3K's downstream effectors and their function
1. AKT/PKB: cell survival
2. Rac: cell migration
65
like Ras, what kinase is also often mutated in cancers
PI3K
66
what is PI3K involved in?
cytoskeleton regulation, cell migration.
its effector Ras involved in actin reorganization
67
different Ras mutations affects different what?
downstream effectors (ex Raf or PI3K)
68
69
what kind of transformation are induced by oncogenes like mutated Ras?
- Increase cell proliferation/division
- Loss of inhibition of cell-cell contacts
- Loss of adhesion
- Increased cell migration
70
how did they test Ras mutant activity?
transfected cells with one Ras mutation or both or none. Culture for 10 days. count the numbers of colonies.
71
what did they find about Ras mutant activity?
each mutation alone was enough to cause oncogenic activity, but the oncogenic activity wwas most when the 2 mutations were present
72
remember: name the 5 subfamilies in the Ras superfamily of small GTPases and their function
Ras: growth and differentiation
Rho: cell morphology and movement
Rab & Arf: vesicular transport
Ran: nuclear import
73
name the main members of the Rho subfamily
Rho, Rac, Cdc42
74
which prenyltransferase post-translationally modifies Rho GTPases again? at what motif?
type 1 geranylgeranyltranserase: CAAX motif where X = Leu
75
what are the specific CAAX motifs of RhoA, Rac1, and Cdc42?
RhoA = CLVL
Rac1 = CLLL
Cdc42 = CVLL
76
how long is the geranylgeranyl group?
20 carbons
77
what is a GDI?
Guanine nucleotide dissociation inhibitor: inhibits GEF
78
GDI has the same effect at what Rac/Rho mutations? meaning it keeps GTPase in what?
N17Rac/N19Rho
keeps the GTPase inactive (GDP-bound)
79
what kind of proteins GEFs?
oncogenic
80
what are the 2 families of GEFs
TRIO (Ddl family) and DOCK
81
what are the domains the TRIO Dbl GEFs family
DH-PH-SH3-DH-PH-SH3-kinase
82
what does the DH domain do?
interacts with GTPase
83
DH domain is specific to what?
the Dbl family (its a Dbl homology domain)
84
what does the PH domain do in GEFs?
localizes the GEF close to PM
85
what are called the domains specific to Rac1/RhoG and RhoA?
GEFD1 is specific to Rac1 and RhoG
GEFD2 is specific to RhoA
86
the GEFD1/2 are made up of what smaller domains?
DH + PH
87
what are the domains that make the DOCK-A Gefs?
SH3-DHR1-DHR2-PXXP
88
what are the DHR1 / DHR2 domains
DHR1 = PH
DHR2 = DH
89
what domain is specific to GAPs?
rhoGAP
90
what domains make up GAPs
SH2, SH3, PH, GAP, PXXP
91
name a few cellular processes controlled by rho proteins (Rho, Rac, Cdc42)
- cytoskeletal dynamics
- cell motility / adhesion
- phagocytosis
- cytokinesis
- apoptosis
- cell proliferation
- cell cycle
92
what is the cytoskeleton involved in?
organelle transport, chromosome segregation at mitosis
93
what processes is actin-based motility involved in? in what way?
- embryogenesis: important morphogenic processes
- inflammatory response: leucocyte migration
- wound healing: fibroblast andvascular endothelial cells migration
- metastasis: tumor cells migration
94
give an example of how actin polymerization allows for cells to move?
unicellular bacteria Listeria moves via its actin tail
95
cell motility involves the coordination of what activities?
cytoskeleton, membrane, and adhesion systems
96
what is the basic engine for gliding and crawling locomotion?
actin cytoskeleton
97
where is actin found in the cell?
monomeric actin filaments are found beneath the PM
98
how microtubules are organized in the cell?
tubulin: rigid structure with one end attached to a centrosome
99
microtubules provide what to the cell?
cell division and cell polarity
100
what do intermediate filaments provide to the cell?
mechanical strenght
101
what is the name of the rope-like fibers made of intermediate filaments?
vimentin and laminin
102
what are the 2 types of actin? describe briefly
- G-actin = monomeric actin bound to ATP
- F-actin: filamentous actin bound to ADP
103
what is special about G-actin's activity?
its an ATPase
104
which type of actin is polymerized?
F-actin!
105
what is the Cc?
when the Koff/Kon = 1 (because filaments maintains a certain length due to equal poly and depolymerization)
106
at which end of the actin filament does polymerization happen?
polymerization is favored at the + barbed end.
depolymerization at the - pointed end
107
elongation of actin filaments only happens when what?
they form and oligomer
108
what molecules are required from actin polymerization?
ATP, Mg2+, K+
109
actin filaments grow in what direction?
toward the barbed + end which points towards the PM
110
what makes that there is more polymerization happening at the pointed + end?
there is more monomeric actin at that end
111
give 2 examples of how actin-binding proteins can act
block actin polymerization by:
- binding to the actin-binding site
- keeping actin ADP-bound
112
name 2 main actin-binding proteins and what they each do
- thymosin: inhibits polymerisation
- profilin: promotes polymerisation
113
what is vinculin/ERM?
actin binding protein that couples actin filament to PM
114
what is Gelsolin?
actin binding protein that breaks the existing F-actin
115
what are filamin and fimbrin?
actin binding proteins thaat link actin filaments
116
what is the Arp2/3 complex?
actin binding protein involved in actin assembly
117
basically name the actin binding proteins that increase actin polymerization
profilin, ARP2/3
118
name the actin binding proteins involved inthe actin filament structure (no dynamic role)
actinin, fimbrin, filamin
119
name the motor proteins that are actin binding proteins
myosins
120
name the motor proteins that are for microtubules
kinesins/dyneins
121
describe the structure of myosin
helical tail that assembles in a dimer with 4 light chains at the neck
122
what are the light chains in myosins?
regulatory subunits
123
myosin II provides what?
contraction and movements of actin filaments
124
how does myosin assemble into filaments? what triggers its activation?
phosphorylation of myosin light chains activates it, releasing its tail causing a spontaneous self-assembly
125
name the 3 types of actin structures and what Rho protein is associated with each
- filopodia: Cdc42
- lamellipodia: Rac
- Stress fibers: Rho
126
what do stress fibers do to the cell? where are they found in the cell
stretch the cell (promote contractility); found everywhere in the cell
127
where are lamellipodia mostly found in the cell and what do they do?
dense mesh of filaments; enriched at the periphery of the cell;
they are involved in cell MIGRATION
128
where are filopodia found in the cell
at the periphery; they extend the membrane
129
what is useful to study actin dynamics in vivo?
toxins!
130
name 3 toxins used to study actin dynamics and how each works
- cytochalisin: prevent actin polymerization by binding to the neg barbed end, allowing depolymerization at pointed end
- phalloidin: binds the side of actin filament and stabilizes them, allowing for visualisation
- latrunculin: stabilizes actin MONOMERS to block their incorporation
131
what method can be used to visualize actin filaments?
indirect immunofluorescence
132
what are the 4 steps of indirect immunofluorescence?
1) fix cells with paraformaldehyde 4%
2) permeabilize with mild detergent
3) incubate with primary antibody and secondary antibody coupled to FLUOROPHORE
4) visualize cell with fluorescent miscroscope with excitation/emission filter
133
how do cells attach to the extracellular matrix?
actin binds to intergin via adhesion complexes / focal adhesion proteins
134
name the focal adhesion proteins
talin, paxillin, vinculin
135
what do integrin bind to on the extracellular side?
fibronectin
136
what activated Cdc42, Rac, and Rho in the context of actin filaments / cell migration?
Cdc42 and Rac = growth factors
Rho = GPCRs
137
what is p160ROCK?
rho-kinase: GTP-Rho binding protein
138
what kind of kinase domain does p160ROCK have?
Ser/Thr kinase domain
139
what domains is p160rock made of?
Kinase domain, Rho-binding domain, PH domain
140
what is p160ROCK involved in?
regulation of smooth muscle contraction, the formation of stress fibers and focal adhesions, neurite retraction, and cytokinesis, etc
141
what domains make up myosin phosphatase?
MBS (myosin binding subunit), catalytic domain, regulatory domain
142
what are the effect of myosin phosphatase?
dephosphorylatesmyosin, deactivating it and causing the disassembly of actin
143
p160ROCK and MBS are therefore believed to regulate what?
the level of MLC phosphorylation cooperatively
144
explain the 3 ways how p160ROCK and Rho-GTP modulates MLC phosphorylation
1) p160ROCK directly phosphorylates MLC
2) p160ROCK phosphorylates MLC phosphatase, inhibiting it
3) RhoGTP directly inhibit MLC phosphate
145
what is WAS?
Wiskott-Aldrich syndrome protein: x-linked immunodeficiency
146
what are the symptoms of WAS?
recurrent infections, hematopoietic malignancies, eczema, thrombocytopenia
147
what is WASP?
arp2/3 activator proteins (no catalytic action, but it is a crosslinker scaffold protein)
148
what does WASP bind to?
Cdc42 GTPase, Arp2/3, F- and G-actin, PIP2, SH3
149
what is the role of the Arp2/3 complex in Listeria?
it activates actin polimerization and bacterial propulsion, and generates new actin filaments
150
how does WASP-N protein get activated and what does it bind to do that?
1) binds PIP2 and Cdc42 -> unfolds and gets activated
2) then binds G-actin and Arp2/3
151
name the 3 rho GTPases, what actin filament structure they form via which adaptor proteins
- Rho -> p160ROCK -> MLC phosphorylation -> STRESS FIBERS
- Cdc42 -> WASP -> Arp2/3 -> FILOPODIA
- Rac -> WAVE -> Arp2/3 -> LAMELLIPODIA
152
in what part of the cell movement are each rho gtpase involved in?
- Rho = stress fibers = back of the cell
- Cdc42 = filopodia = cell direction/middle
- Rac = lamellipodia = forward/cell adhesion
153
in what order do the rhogtpases act in cell movement?
1) Cdc42 (cell direction movement)
2) Rac (protrusions)
3) Rho (contraction)
154
how did they do the 2D migration assay?
- stain F-actin
- stimulate cells with growth factor (rac), GPCR (RhoA), bradykinin (Cdc42)
- express a GAP in the cells and see which cells stop moving
155
what did they find when using dominant negative GTPases as a tool to determine their functions in signaling pathways?
Dominant negative RacN17 but not Cdc42N17 blocks
The ability of the receptor to induce lamellipodia formation
156
explain the GST-pulldown Rho GTPase Activation Assay
- added a GTPase specific effector bound to GST
- gluthatione beads pulldown
- western blot with Cdc42 and UNC5 (receptor) antibodies
157
when they added the ligand netrin (UNC5 ligand) what did they find?
increase in Cdc42 -> UNC5 activation can modulate Cdc42
158
what is ICY?
FRET: intermolecular CFP/YFP reporters
159
using FRET/CFP, they found that UNC5 does what?
activate RhoA in cells with neurites
160
basically what are UNC5 receptor activation's effects on neurite, and it acts via what protein?
UNC5 activates Rac1 -> induces neurite formation and extension.
UNC5 activates RhoA -> induces neurite stability
