Unit 2 Chapter 4, 5,6 Flashcards
Kinases
are enzymes that catalyze the
transfer of gamma phosphate group from
ATP/GTP to hydroxyl groups on a specific
amino acid in a target group
Phosphatase
an enzyme that removes
phosphate group from a specific amino acid in a
target group
EGFR’s belong to a family of
receptor tyrosine kinases
EGFR include 4 types of receptors
ErbB1 (HER1),
ErbB2(HER2)
ErbB3 (HER 3)
ErbB4 (HER4
Steps of EGF signaling
- Binding the growth factor to the receptor
- Receptor dimerization
- Autophosphorylation
- Activation of intracellular transducers
- A cascade of serine/ threonine kinases
- Regulation of transcription factors and gene expression
Steps 1 and 2 of EGF binding and dimerization
EGF binds to domain 1 and 2, Domain 3 binding to EGF
This exposes domain 2 and forms a dimer with another EGF bound to EGFR
Autophosphorylation in EGF/EGFR
Dimerization enables the kinase domains of one receptor to phosphorylate the other receptor and vice versa
▪ The change in receptor conformation permits access to ATP and substrate to the catalytic kinase domain
Why is autophosphorylation crucial in EGF/EGFR?
it’s crucial for recruitment of cytoplasmic proteins
RAS
responsible for integrating growth factor signals
from membrane to nucleus
Describe ras
GTP-binding proteins and are activated when
bound to GTP and inactivated when bound to GDP
RAS-GTP binds to and contributes to
the activation
serine/threonine kinase RAF
Describe the RAS to MAP Kinase signaling cascade
Ras-GTP Binds to activation of RAF
Activated RAF phosphorylates MEK
Activated MEK Phosphorylates MAP Kinase
MAPK affects activity of trancription factors by phosphorylation
Transcription Factor
group of proteins that bind to DNA and regulate
the expression of genes involved in growth, differentiation and death
AP-1 gene transcription factor is target of
Map Kinase
AP-1 gene products activate
cyclin D genes which are critical regulators of cell cycle
Star player in regulating growth
RAS
Describe RAS activation of AKT
Ras interacts with PI3K by activating PIP 2 to PIP 3
PIP3 recruits PDK-1 and AKT to the membrane
AKT translocates to the nucleus, phosphprylates nuclear transcription factors
AKT is involved in
anti-apoptotic and survival roles
In normal cells, activated RAS is
is inactivated
immediately by GAPS protein
inactivated RAS causes
conformational change in RAS and releases RAF which then is inactivated by phosphatase
Inactivation of RAS
Brings thecell back to normal non-proliferative state
In cancer cells RAS can be
continuously activated by mutations induced by various factors
Mitated RAS cannot be inactivated by GAPS protein as in normal cells
this results in continuous proliferation of tumor cell
Growth factor signaling not only leads to cell
proliferation but also can effect
cell behaviors such as adhesion and motility
SRC
an intracellular tyrosine kinase coded by genes
src
SRC plays an important role in
cell adhesion, invasion and motility upon EGF activation (Metastasis)
Upon stimulation of EGF receptor by growth factor
the autophosphorylated receptor can interact with SH2
domain of SRC and activate the protein
One way in which SRC can be activated is
via EGF receptor
Activated SRC activates
wide range of target proteins such as focal adhesion proteins (FAK), adaptor proteins, motility proteins and transcription factors (Important in metastasis)
What pathway needs to be targeted to limit metastasis and stop FAK protein? (DESCRIBE)
EGF-EGFR-> Autophosphorylation Inactive SRC Active SRC FAK Increased motility and cell invasion (This pathway needs to be targeted to limit metastasis and stop FAK protein)
Monoclonal antibodies that block the signaling pathway prevents growth factor from binding to the receptor
Herceptin
Erbitux
Vecitibix
Monoclonal antibodies that block the intracellular domain autophosphorylation stopping
Gilotrif
Tarceva
Tykerb
Monoclonal antibody that shuts down things going into the nucleus
Mekinist (affects MEK)
Monoclonal antibodies that shut down RAF signaling
Nexavar
Zelforab
Tafinlar
Herceptin (trastuzumab)
a monoclonal antibody that binds the extracellular domains of ErB2 receptor with high affinity
Herceptin functions
through a combination of mechanisms including enhanced receptor degradation, inhibition of angiogenesis, cell proliferation and recruitment of immune cells, resulting in antibody-dependent cellular cytotoxicity
Erbitux has 5 mechanisms of action
Cell cycle arrest of cancer cell and inhibits proliferation, prevents metastasis inhibits angiogenesis antibody-dependent cellular cytotoxicity inhibits DNA repair mechanisms
ressa and Tarceva
small molecule kinase inhibitors that are directed against tyrosine kinase activity of EGFR family members
Imatinib (Gleevec) used for
Chronic myelogenous leukemia
(CML) accounts for 15-20% leukemias
Most CML patients carry
Philadelphia chromosome chromosome generating BCR-ABL fusion protein
Gleevec
a tyrosine kinase inhibitor that binds to ATPbinding pocket within catalytic domain and inhibits the
action of enzyme
proto-oncogenes
Normal cellular genes that can be converted to
oncogenes
RAS proto-oncogene
normal gene produces normal protein that controls proliferation
Mutations in RAS proto-oncogene
can transform it in to RAS oncogene which can cause cancers due to uncontrolled proliferation
Oncogenes
genes whose presence can contribute to uncontrolled cell proliferation and cancer
Oncogenes contain which type of mutation (Dominant/recessive)
Dominant
More than ___ oncogenes have been identified and
proteins they produce fall into different categories
100
Most proteins produced by oncogenes are
components of signaling pathways that promote cell
proliferation and survival
B-RAF Function, Protein, and associated disease
Intracelluar signal transducers
Protein-RAS
Melanoma
Myc Function, Protein, and associated disease
c-myc (Proto-oncogene)
Transcription Factors
Myc Protein
Burkitt’s Lymphoma
RAS Function, Protein, and associated disease
Intracellular signal transducers
RAS protein
Bladder and lung cancer
V-erbB
C-erbB (Proto-oncogene)
Growth Factor Receptor
EGFR (Receptor)
Breast Cancer
Chromosomal translocation
Part of the chromosome is mixed with another part of
another chromosome, altering promoter region and
amount of protein created
Gene amplification
One gene has multiple copies and is amplified in function. Elephants have multiple p53 genes compared to humans
Point Mutation
Changes a single nucleotide in the DNA sequence, changes mRNA, changes AA sequence, and changes the protein or conformation of the protein
Cellular oncogenes arise due to the following
mechanisms (convert from proto to oncogene)
Point mutations Gene amplification Chromosomal translocation DNA Rearrangement Insertional Mutations
Single mutation in RAS proto-oncogene results in
RASoncogene that produces mutated Ras protein in which single amino acid is converted from glycine to valine
Neuroblastoma with extensively amplified MYC gene are more likely to
invade and metastasize and lower survival
rates (associated with adrenal glands)
(Map Kinase applies here)
Philadelphia chromosome
abnormal version of chromosome 22 in 90% of all cases of chronic myelogenous leukemia
Translocation of ABL gene (tyrosine kinase) in
chromosome 9 to BCR gene (Serine/threonine kinase) on chromosome 22 and results in
abnormal BCR-ABL fusion gene and fusion protein
Viruses have special viral sequences at its end called
LTR’s (Long terminal repeats) which promote gene expression
How long does mitosis take?
16 hrs
How long does binary fission take?
24 mins
Cell Cycle Steps
G1 (Growth) S Phase (Synthesis) G2 (Growth) M Phase (Mitotic) Cytokinesis (Cytoplasm split)
Interphase
G1
S
G2
Proteins involved in eukaryotic DNA Synthesis
DNA polymerase Helicase Topoisomerase SSBPS Ligase RNA Polymerase
Differentiated, living cells are also called
quiescent
G0 Phase
Outside the cell cycle
Cell is non-dividing and differentiated
dependent on growth signals for division
G1 Restirciton Point
Control point in cell division
If a cell crosses it’s point, it becomes irreversibly committed to go through the cell cycle without growth factors
Cyclins
proteins that have a critical role in cell cycle
coordinate and regulate the passage of
cell through different phases of cell cycle
Cyclins act as
regulatory subunits of cyclindependent kinase (cdks).
Upon binding of cyclin to cdk partner, cyclin undergoes
a conformational change in the catalytic domain, exposing an active site.
Concentration of cyclins during cell division is
dependent on
Transcription of cyclin genes
Regulated protein degredation
Cdk proteins are regulated by
binding of cyclins.
Does S Phase have a checkpoint?
NO
Cyclin E and CDK2 role in the cell cycle
Takes the cell into S Phase
CyclinD and CDK 4 and 6 role in the cell cycle
Push the cell over the restricition point, cross through the checkpoint
Mitogen
small bioactive protein or peptide that induces a cell to begin cell division, or enhances the rate of division (mitosis).
Mitogenesis
the induction (triggering) of mitosis, typically via a mitogen.
Mitogenesis
the induction (triggering) of mitosis, typically via a mitogen.
What phases of the cell cycle have checkpoints?
G1
G2
M
G2 Checkpoint
Check to make sure cell is ready for mitosis, no mutations in DNA, no breaks in chromosomes, all microtubules ready for mitosis
Cyclin A and CDk2 role in the cell cycle
Help push through the S phase
Cyclin D
The first cyclin to be synthesized, and together with CDK 4/6 drives progression through the G1 Restriction point and G1 Checkpoint
Cyclin D regulates
Expression of Cyclin E, which binds to CDK2 and is required for G1 to S phase transition
CyclinA-CDK2 is important for
S phase progression
Cyclin B/A-CDK1 Directs
G2 and G2 to M Phase Transition
Cyclin order
DEABA
CDK Order
4 2 1
Why are cell cyle checkpoints important
Maintaining the integrity of the genome
G1 Checkpoint
Arrest of the cell cycle in response to DNA Damage
G2 Checkpoint
arrest of cell cycle in response to DNA damage and/or unreplicated DNA to ensure proper
completion of S phase
M Checkpoint
arrest of chromosomal segregation in response to misalignment on the mitotic spindle
Disruption of checkpoint leads to
Mutation and carcinogenesis
Function of cyclin-CDK Complexes
They exert their effect by phosphorylating target
proteins
Target proteins of Cyclin-CDK complexes
Histones
▪ Transcriptional regulators
▪ Cytoskeletal proteins
▪ Nuclear pore proteins
Cyclin-CDK protein interaction with proteins results in
▪ Regulated gene expression
▪ Mitotic spindle assembly
▪ Chromosomal condensation
▪ Nuclear membrane breakdown
CDKs
serine/threonine kinases that,
sequentially, regulate progression through the
phases of the cell cycle via phosphorylation
Four Mechasnisms of CDKs
▪ Association with cyclins
▪ Association with cdk inhibitors
▪ Addition of phosphate groups to activate cdk activity
▪ Addition of phosphate group that inhibits cdk activity
binding of cyclins to their partner cdk causes
a crucial conformational change in the cdk that
allows
the binding of protein substrates and correct
positioning of ATP
Degradation of cyclin proteins is carried out by
proteasome through ubiquitin, which flags the
cyclin protein for degradation
P16 binds with
cdk 4/6 and interferes with the binding of cyclin D to cdk4/6.
P21 inhibitor binds to
both cyclin E and cdk 2 blocking ATP –binding site, thus disabling kinase activity.
CDK inhibitors are regulated by
ubiquitin-mediated degradation.
Addition of the phosphate group to the threonine 14 and 15 positions by wee 1 Kinase
inactivates the cyclin
Removal of the phosphate groups to threonine 14 and 15 positions by CDC 25 Phosphatase
Activates the Cyclin
Function transcription factors
Proteins that control transcription
E2F Makes
SSBP DNA Polymerase Primase Helicase Ligase
When Rb (Retinoblastoma) protein is binded to E2F
Genes needed for S Phase are NOT Transcribed
When Rb protein is phosphorylated and unbound from E2F,
E2F is free to promote transcription
MRNA Translation
Enzymes and other proteins required for S phase
Cell Proliferation
Rb Protein serves as
a molecular link for the G1-S phase transition
Rb binds to transcription factor E2F which is
crucial for the expression of genes needed for S
phase.
Rb protein comprises the
A domain and B domain joined by a linker region
Histone deacetylase (HDAC) binds to
domain B and E2F binds to domain A
Function of HDAC
wraps the DNA tightly around the Histone, increasing positive charge of histone and it glues more to the DNA
Hypophosphorylated Rb
sequesters E2F/DP and HDAC which results in complete repression of transcription.
Partial phosphorylation of Rb protein by cyclin D/cdk4/6 causes
a conformation change releases HDAC but not E2F/DP. This results in relieving repression of some genes such as cyclin E but not E2F target genes
Additional phosphorylation by cyclin E-cdk2
causes
an additional conformational change and releases E2F/DP, and transcription of all of E2F target genes.
The G2 checkpoint
blocks entry into M phase in cells that have incurred DNA damage in previous phase or have not correctly completed S phase
DNA damage activates
ATM or ATR. These kinases then phosphorylate and activate chk1 and chk2 kinases
One target of the checkpoint kinases are the cdc25
tyrosine phosphatase
regulate cdk activity by removing inhibitory phosphatase
Activation of G2 checkpoint results in
the inhibition of cdc25 by chk1.
decatenation
the process of separating this physical linkage
Z-TMS
induces cell-cycle arrest at G2/M Phase
that is accompanied by the activation of p21, downregulation of CDK1
Cleavage Furrow
Contracting ring of microfilaments
Mitotic Checkpoint is also known as the
spindle assembly checkpoint
Mitotic checkpoint prevents
mis-segregation of chromosomes
If any of the sister chromatids are not attached to
microtubules at their centromeres during mitosis
they recruit checkpoint proteins that act as inhibitors of
anaphase-promoting complex such as securin
When securin are attached to sister chromatids without microtubules
they inhibit enzyme separase
Separase
cleaves the link (cohesin) between sister
chromatids and helps their separation during anaphase
Aurora Kinase
regulate important aspects of mitosis, such as
chromosome segregation and the spindle
checkpoint
Mutations in cell cycle regulators can result in
aberrant regulation of cell cycle, uncontrolled proliferation and carcinogenesis
Chromosomal translocations cause over-expression
of
CDK6 in some leukemia’s
DNA amplification of cyclin D and E by gene
amplification result in
15% of breast cancers and 20% squamous cell carcinoma
p16 inhibitor deletions have been observed
Pancreatic cancers
Abnormal chromosome numbers or aneuploidy
caused by
defects in centrosomes, mitotic spindle,
or cytokinesis is often observed in many solid
tumors
Over expression of Aurora kinase A gene has
been commonly reported in
94% of invasive ductal breast adenocarcinomas
Flavopiridol acts as
competitive inhibitor of all cdks.
▪ It induces cell cycle arrest at G1/S and G2/M phase.
▪ It also regulates gene expression of cyclin D1 and D3.
CYC-116 and AZD1152
Are aurora Kinase inhibitors
Vinblastine
inhibits microtubule assembly resulting in activation of mitotic checkpoint
Paclitaxel/taxol
Mitotic inhibitor
Chemotherapeutic effects on the GI Tract
Ulcers, nausea, vomiting, diarrhea, loss
of appetite
Chemotherapeutic effects on the Bone Marrow
Suppressed immune system, anemia
Chemotherapeutic effects on the skin
Skin easily damaged, wounds take longer
to heal
chemotherapeutic effects on Hair follicles
Hair Loss
Necrosis
Cells swell, membranes become leaky, and the cells spill out their contents into the surrounding tissue and cause inflammation
Autophagy
A type of cell death that is self-eating
Autophagosomes
spherical structure with double layer membranes. It is the key structure in macroautophagy, the intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles, invading microorganisms)
Steps of autophagy
- Starts with ER and formation of preautophagosome
* Matures into autophagosomes and fuses with lysosomes in cells
Activation signals for autophagy
○ Triggered by starvation
○ Cellular Stress
○ Infection
Proteins invovled in autophagy
LC3, Beclin-1 ULK
Autophagy is inhibited by
○ Cell growth and proliferation
○ Excess cellular energy
○ MTOR and PI3K complex
Apoptosis
- Active cell death that happens in normal cells and diseased organs
- Need ATP For this process
- Involves a single cell
- Programmed Cell Death (Basically from a checklist
- Decrease in cell size
- Cell membrane is intact
- Does not trigger local inflammatory responses or absent
- Involves cellular Makers CD95 protein
- DNA Gel has a ladder pattern
Necrosis
• Passive Form of Cell death that happens in injured tissue or organs
• No ATP Needed
• Involves groups of cells
• Involves Extrinsic (External) stimuli
• Increase in Cell size (Cells swell and Explode)
• Cell membrane damage is involved
• Involves local inflammatory response (Activating immune cells
• in the local environment
• No Cellular Markers (G1 Phase Cyclins, and things like that)
DNA has a smear pattern
Apoptosis
Highly regulated programmed cell death
Apoptosis plays an important role in
developmental morphogenesis
Control of cell numbers and tissues
Gets rid of damaged cells
Apoptosis is a main Tumor suppressive mechanism that
Disposes the cell with extenisve DNA damage and mutations
A specialized group of proteases called _________ play a main role in apoptosis
Caspases
Proteases
enzyme that breaks down proteins
Caspases
Specialized proteases involved in apoptosis
Annexin V
A phospholipid binding protein
Caspases
Cysteine-Rich Aspartate Proteases
How many mammalian caspases have been identified
13
Caspases are synthesized as inactive ________ which when cleaved as aspartate residues results in activation of enzymes(_______)
Procaspases
Caspases
Caspases participate in a cascade of activation , activating
downstream caspases and amplifying the signals
Cells may be induced to undergo apoptosis by what external factors
Extracellular signals
TNF (Soluble) death factor
Membrane-bound fas ligand bound to neighboring cells or certain immune cells
Cells may be induced to undergo apoptosis by what internal factors
DNA Damage or oxidative Damage (Ionizinf radiation-Reactive Oxygen Species
If CDC 25 is active or not inhibited by CHK1
CDC25 dephosphorylates CDK protein
CDK activates
Progression of cells into Mitosis phase
If CDC25 is inactivatedby CHK1
No dephosphorylation of CDK protein
No progression of cells into M Phase
Cell stress signals and DNA damage activate
ATM/ATR kinases
which activate CHK1 / Casein Kinase II
These disrupt the P53-MDM2 Complex which activates P53 Protein
P53-MDM2 complex activates
P53 protein
P 53 activates ____ Protein, which in turn activates ____ Protein
Bax
Bid
Conormational change in BAX protein causes it to
insert into the outer mitochondrial membrane and oligomerize (6-8 molecules)
Mitochondria release
Cytochrome C and Procaspase 9 into the cytoplasm
Released cytochrome C and procaspase 9 bind with
apaf-1 to form the apoptosome
caspase aggregation leads to
activation of procaspase 9
which in turn triggers caspase cascade activating caspase 3
Caspase 3 cleaves
target proteins and causes apoptosis of the cell
Anit-Apoptotic proteins
BCL-2
BCL-X
BCL-W
Pro-Apoptotic Proteins
BAX
BAD
BID
Extrinsic pathway mediated by membrane death receptors
TNF and FAS bind to their receptors
Binding causes conformational change and oligomerization
Adaptor proteins TRADD and FADD recognize the activated receptors and lead to the aggregation of Procaspase 8
Procaspase 8 aggregation leads to the activation of caspase 8
Caspase 8 initiates a caspase cascade, proteolysis and apoptosis
Both intrinsic and extrinsic pathways activate
Caspase 3
______ is an important mediator of apoptosis as it moves the cell into execution phase of apoptosis
Caspase 3
_____ causes proteolysis of different target proteins
Caspase 3
Target proteins for execution of apoptosis
Nuclear Lamins
Cytoskeletal proteins
Activation of DNAse
Nuclear Lamins role in apoptosis
Allowing for nuclear membrane shrinkage
Cytoskeletal proteins’ role in apoptosis
such as actin filaments affecting the cell structure
Activation of DNAse role in apoptosis
Resulting in cleavage of DNA
The extrinsic pathway of apoptosis is regulated by inhibitor protein
CFlip
C-Flip binds to _____ and inhibits What?
FADD
activation of activation of procaspase 8
Inhibitors of Apoptosis (IAPs) regulate apoptosis by
binding and inhibiting the activity of caspase 3 and caspase 7
SMAC (Second Mitochondria-Derived Activator)/ DIABLO
Is released from mitochondria eliminates the inhibition by IAPs
Caspase 8 is a key regulator of
Extrinsic pathway and also cleaves and activates BID in the intrinsic pathway
How do intrinsic and extrinsic pathways of apoptosis converge
Activation of caspase 3, which causes proteolysis
Cancer cells contain
activated caspases that are inhibited by upregulated IAPs
TRAIL Receptors
triggers apoptosis via extrinsic pathway
TRAIL receptor and TRAIL Ligand interactions induce
apoptosis in many cancer cells but not in most normal cells
Mutations in death receptor genes such as FAS and Trail have been reported in what types of cancers
Melanomas and squamous cell carcinoma
Suppression of Caspase gene expression has been demonstrated in
small-cell lung carcinoma
mutation in LEU62
Blocks the interaction of caspase 8 with the adaptor FADD, thus stopping the signal
Mutations in p53 genes
Provide the cancer cells with a survival advantage by disrupting apoptosis is common in lymphomas
Chromosomal translocation of ___________is observed by many B-Cell lymphomas
anti-apoptotic protein BCL2
Mutations in ___ and ___ genes that code for apoptotic proteins are mutated in______
Bax
BID
50% of colon tumors
A successful chemotherapy will be one that
triggers apoptosis
Many Chemotherapeutic agents
trigger DNA mutations or DNA damage, thus activating the intrinsic apoptotic pathway
What contributes to chemotherapy resistance
Upregulation of anti-apoptotic members of BCL2 family and the downregulation of pro-apoptotic members
