D103 Final Review

signal transduction importance

involved in development → issues can affect development and tissue homeostasis

signaling pathway composition

stimulus (intra or extra-cellular)

receptor

transduction cascade

effector

responses

stimulus aka ligand

hydrophilic molecules require transmembrane receptor

hydrophobic molecules require intracellular receptor

ligand-receptor interaction should have high specificity but low affinity → REVERSIBLE!

overall, should be made, mobilized, altered/degraded rapidly

receptor

ligand gated ion channel

gpcrs → use a GTPase to transduce signals

enzyme coupled receptors (rtks) → use a kinase to transduce signals

transduction cascade

mediated by proteins → pass a signal downstream

control them by altering activity → ie modifications like phosphorylating to inhibit/activate

how do intracellular components of a transduction cascade pass a signal?

want to induce a reversible protein conformational change

can have p-p interactions, ion-p interactions, GTP-effector interactions, or reversible modifications such as ubiquitination

protein domain

region of aas that can fold independently of the rest of the protein

serves as a point of recognition → mediates interactions between proteins

ex) sh2 domain binds to phosphorylated tyrosine residues in proteins

protein motif

conserved subset of aas within a domain, essential for domain function

domain role in signaling pathways

mediate interactions between intracellular signaling proteins

recruit pathway components and send signal downstream

ex) rtk has p-tyrosine sites on tail, can be recognized by sh2 domain on protein a → protein a can be recognized by the domain of protein 2 → etc etc

signal transduction complexity

one signal can activate multiple pathways

these pathways can share components

can be linear, convergent, or have multiple branches

contact-dependent signaling

signal cell has a membrane bound signal molecule, gets recognized by receptor on target cell

paracrine signaling

ligand released from signaling cell to neighboring target cell

synaptic signaling

neurotransmitter released from neuron, travels axon to synapse

endocrine signaling

hormones sent through blood to target tissue

NO → intracellular signaling

from l-arginine by NOS (endothelial NOS is in blood vessels)

• regulated by Ca2+-calmodulin complex

ACh binds to GPCR → IP3 produced → IP3 heads to ER → Ca2+ release → calmodulin activation → NO production

NO heads to epithelial cell → guanylyl cyclase activated → GTP converted to cGMP → protein kinase G activated → myosin phosphorylation inhibited → vasodilation

angina treatment

angina → insufficient blood supply to heart

nitroglycerin can be broken down to NO

viagra inhibits cGMP phosphodiesterase → increases cGMP levels

steroid → intracellular signaling

steroids are v hydrophobic molecules

generally bound to plasma protein carriers, only free steroid hormones can participate in signaling transduction

receptors can be in cytoplasm or nucleus

• also have some membrane bound receptors that are enyzme coupled and typically have rapid response

steroids bind to cytoplasmic receptors then get translocated into nucleus, or bind to nuclear receptors from the get go

• translocation of ligand-receptor complex occurs bc the interaction causes a conformation change, revealing the NLS

steroid receptor complex then has transcription factor activity and can produce new proteins

• slower than membrane bound

nuclear hormone receptors

have a variable region, a DNA binding domain, and a ligand binding domain

can bind to DNA and act as transcription factors

also located on inner leaflet of pm

activation of hormone receptors

can have basal condition bound to repressor complex

binding of ligand induces conformational change, loss of repressor and association of activator

androgen insensitivity syndrome

dysfunctional androgen receptor AR cannot recognize ligand

body tries to compensate by upping ligand levels, but AR cannot recognize it

ligand is converted by protein to other hormone (testosterone -aromatase-estrogen)

anabolic steroids

increase muscle and bone synthesis

gpcrs

G protein is a type of GTPase → activated by GEF and deactivated by GAP

three subunits acting together → alpha, beta, and gamma

• alpha subunit has the GTPase activity

• a and y have lipid anchors

allosteric binding increases affinity of primary binding site for target

• ca2+, ATP, GTP can act as allosteric factors

• allosteric binding causes conformational change in GTPase

have 7 transmembrane domains

stimulation of alpha subunit to exchange GDP → GTP

• so ligand binding has GEF activity

effector is typically an enzyme

gpcr signaling

ligand binding causes association with heterotrimer

activation causes alpha subunit to change shape and triggers dissociation of GDP

alpha subunit then binds GTP, dissociates from b/y subunits

alpha subunit now has binding site for target effector, activating it

binding leads to hydrolysis of GTP to GDP, leads to dissociation of alpha subunit

• thus effector has GAP activity

GTP-alpha then reassociates with b/y subunits

in general":

1. ligand-receptor

2. receptor-Ga

3. Ga-effector

common gpcr effectors

adenylyl cyclase

phospholipase C

cGMP phosphodiesterase

K+ ion channel

b/y subunits

can also activate ion channels

ultimately, alpha subunit still controls length of response → signal turns off upon GDP exchange

• this is because the GTP exchange leads to b/y unit dissociation

gpcr inactivation

gpcr kinase/grk can phosphorylate the gpcr domain and recruit arrestins to turn off signaling

1. arrestins prevent interaction with other downstream effectors

2. act as adaptor proteins for clathrin → endocytosis

receptor sequestration

receptor is temporarily internalized by endosome

receptor down-regulation

internalized and destroyed in lysosomes

receptor inactivation

modified so that it is unable to interact with g protein

ex) phosphorylation

second messenger

small signalling molecules that transduce the signal response

ex) ca2+, IP3, DAG

phospholipase C

second messenger enzyme

generates DAG and IP3 from PIP2

• DAG → hydrophobic, remains on membrane

• IP3 → hydrophilic, dissolves rapidly into cytosol

remember Ca2+-calmodulin?? yeah this is it

IP3 will bind to receptor on ER, released Ca2+

Ca2+ then activates protein kinase C

DAG can also recruit and activate pkc

leads to phosphorylation of various protein residues

phospholipase c inactivation

IP3 can be dephosphorylated or phosphorylated to IP4/IP2 which don’t have activity

DAG can also be phosphorylated

adenylyl cyclase

second messenger enzyme

ATP → cAMP → protein kinase a activation → CREB phosphorylation and activation → gene transcription

• note, cAMP is quickly broken down by cAMP phosphodiesterase

• cAMP binds to regulatory subunits, causing a conformation change and subsequent release of active catalytic subunits

cell surface receptors

all transmembrane proteins

single pass, aka one domain

either have intrinsic enzyme activity, or associate with one

rapid or slow responses

associated with development and cancer

cell surface receptor activation

monomeric receptors dimerize with one another after binding of ligand

can also have dimers that undergo a conformational change after ligand binding

alternatively, dimerization can be initiated by monomeric ligand clusters on neighbor cells

dimers can phosphorylate and activate eo

tgf-b pathway

ligand: tgf-b

receptors: tetramer → two of each type 1,2 + sometimes 3

• have intrinsic kinase activity

transduction proteins: Smad

• R-Smad (receptor regulated)

• Co-Smad

• I-Smad (inhibitory)

ligand binds receptor 2/RII

RII interacts with and phosphorylates RI

activated RI has increased affinity for R-Smad

RI phosphorylated R-Smad, conformational change exposes NLS

phosphorylated R-Smad forms trimeric complex with another R-Smad and Co-Smad

trimer gets bound by importin and initiates translocation to nucleus

GTP-Ran then undocks trimer from importin

trimer binds w/another protein to form transcriptional activation complex

net result is gene transcription

tgf-b inactivation

ski protein

• disrupts trimer formation, preventing translocation into nucleus and formation of transcription complex

• also recruits repressors to the gene

• ski expression is induced by tgf-b signaling, is negative feedback

I-Smad

• blocks RI from binding R-Smad, no activated R-Smad3 and thus no trimer formation into nucleus

• can also dephosphorylate RI and R-Smad

cytokine signaling pathway

ligand: cytokines → hormones and growth factors

receptors: 2x cytokine receptors

• associated with JAK, a tyrosine kinase

transduction protein: STAT proteins

ligand binding induces receptor dimerization

one jak phosphorylates the other, activating it

jak then phosphorylates the receptor tails, leading to recruitment and phosphorylation of STATS

• their SH2 domains recognized the p-tyrosine

STAT phosphorylation leads to dimerization, exposing NLS

STAT dimers then get translocated to nucleus and activate transcription

cytokine inactivation

proteins can bind to receptor and jaks by SH2 domains

• can have SHP1 dephosphorylate them

• or SOCS/an e3 ligase ubiquinate them (ads ub to mark for degradation)

Ras-MAPK signaling pathway

ras is a v important oncogene, a mutation can accelerate the cell cycle and cause uncontrolled proliferation

ligand: soluble/membrane-bound protein

receptors: rtks

ligand binding causes dimer to undergo conformational change → tails phosphorylate eo and add even more tyrosine residues

cross phosphorylation generates new binding sites for downstream components

scaffolding function, associates grb2 → associates SOS (GEF) → associates Ras GTPase → Ras activation → downstream effectors in MAPK pathway

• Raf

• Mek

• Erk

cascade of serine/threonine kinases results in cell cycle control

dominant positive rtk mutation

receptor constitutively on, ligand independent signaling

dominant negative rtk mutation

dimer cannot cross phosphorylate, kinase constitutively off

best way to control signaling?

modification of second messengers

all-or-nothing binary response

choice between two cell fates, involve positive feedback

hyperbolic responses

fine tune metabolic processes controlled by hormones

sigmoidal responses

most responses

reduce inappropriate responses to bg level of signal

cells need to think, differentiates between true and false stimulus

• ex allosteric binding

positive feedback

transient stimulation is sufficient to generate sustained activity, even after stimulus removal

negative feedback short delay

limits level of response, one peak then kinase is at higher levels than baseline

negative feedback long delay

limits level of response, kinase is able to recover so there’s an oscillatory response

signaling in a noisy environment

true signal output must be robust and sustained

must also have binding specificity

• surrounding residues can help with specificity

• as can scaffold proteins

signaling pathway crosstalk

components can have both shared and unique properties within a pathway

key events of cell division

increase cell number/produce more tissue

make an accurate genome copy

maintain DNA → segregate duplicated genome to each daughter cell

maintain organelles → segregate organelles to each daughter cell

interphase

cells grow and replicate DNA

G1

S → DNA synthesis

G2

• gap phases: key cellular activity to prepare for s and m phases

cell cycle phase length

50% in G1 phase

m is usually shortest

early embryogenesis

onlu s and m

cell size decreases, no g phases to synthesize building blocks

cyclin/cdk

regulate cell cycle

cdk is cyclin dependent kinase

• stable concentration

cyclin concentration fluctuates between cycle phases

• cdk cofactor, activates cdk

• recruits substrates for cdk

coordinate cell cycle events by phosphorylating multiple substrate proteins

g1-cyclin

controls the activity of g1/s and s cyclin

g1/s cyclin

activates cdk in g1, prepares for s phase entry

s cyclin

binds cdk at start of s phase, stimulates chromosome duplication

m cyclin

activates cdk at g2/m transition state for m entry

s and m cyclin must be degraded to complete mitosis

cyclin/cdk activation

t-loop originally blocks active site

cyclin binding causes conformational change, exposes active site

cak phosphorylation changes t loop shape, fully activating cdk

cyclin/cdk inhibition

wee1 phosphorylates complex and deactivates it

• cdc25 phosphatase dephosphorylates it

inhibitory proteins can also bind and inhibit the complex

cyclins themselves can be targeted for ubiquitination and degradation

• apc/c is an e3 ligase

g0

differentiated cells that dont undergo cell division

can re-enter cell cycle given appropriate stimulus

• growth factors and mitogens stimulated by wounds can cause re-entry

growth factors

stimulate cell growth, protein synthesis, and nutrient uptake

bind to rtks

lead to increased ribosome production

ultimately increased protein synthesis and cell growth

mitogens

stimulate cell cycle entry

activate ras-mapk signaling pathway

leads to myc synthesis

myc increases g1-cyclin → g1/cdk activated

Rb protein inactivated by g1/cdk

• rb is an inhibitory for e2f

e2f promotes cell cycle genes ex) g1/s and s cyclins

positive feedback

rb protein

negative regulator of g1 phase

inhibits e2f dependent expression of genes required for s phase entry

key tumor suppressor

cell cycle checkpoints

block movement into the next cell cycle under abnormal conditions

function by controlling cyclin/cdk activity

can arrest cell cycle at checkpoints until conditions are met for resumption of cycle

g1/s checkpoint

dna damage can induce synthesis of cki (cdk inhibitors)

cell cycle arressted until dna repair completed, or until cell death

can be lost in cancer cells

ex p53 induces cki gene expression

s phase

regulated by s-cyclin/cdk

replication origins are bound by orc

cdc6 associates with orc and dna helicase to determine site of dna synthesis

s-cyclin/cdk phosphorylate proteins that activate dna rep

also inhibit orc/cdc6 to ensure only one round of dna rep

• dont want multiple replication origins

entry to m phase sees to the destruction of s-cyclin/cdk

• also phosphorylated orc/cdc6 to allow function for the next cycle

g2 phase arrest

damaged dna activates g2 checkpoint

inactivates m-cyclin/cdk by cki

mitosis

cell segregates and duplicated chromosomes move to opposite ends of spindle

role of m-cyclin

m-cyclin is required for mitotic progression

it must be degraded to allow for mitotic completion

• ex by apc/c during late mitosis

  • +cdc20 to degrade at end of anaphase

  • +cdh21 to degrade during late mitotsis and early g1

budding yeast

can analyze g1/s transition

cell morphology is v distinct between g1 and s

one cell to one budding cell

fission yeast

can analyze g2/m transition

cell morphology is v distinct, start pulling apart between g2/m

yeast model system

v useful bc haploid, genotype translates directly to phenotype

ts mutatnts

temperature dependent lof mutation

make two identical plates and incubate at permissive and restrictive temperatures

• restrictive temps will prevent the mutated gene from functioning, so it will arrest in the phase where the gene is essential

facs

fluorescence activated cell sorting

s phase cells will have more dna than g1 phase

g2/m cells will have twice the dna as s phase

identifaction of cdk mutations

yeast cDNA library → each plasmid has a yeast gene

random uptake of a plasmid

perform rescue experiment at restrictive temp

• any colony that can grow has rescued functions

• can identify which gene was uptaken

cdc2 and cdc13

mutants delay or fail to enter mitosis

2 encodes cdk1

13 encodes m-cyclin

wee1 mutant

premature mitosis entry

mutant prevents inhibition of cyclin-cdk complex, leads to net activation

cdc25 mutant

delayed mitosis entry

mutant leads to net inhibition of cyclin/cdk complex

positive feedback of m-cycln/cdk

activates cdc25 and inhibits wee1

mitosis challenges

no new protein synthesis after entry

condense chromosomal dna before separation

nuclear envelope disassembly

sister chromatid sorting

mitosis events

6 total

1. prophase: separation of centrosomes and sister chromatid condensation

2. prometaphase: nuclear envelope removed, chromosome capture → spindle forms and aligns

3. metaphase: chromosomes are aligned on plate

4. anaphase a: sister chromatids move to opposite ends of cell

5. anaphase b: cell elongates via spindle elongation

6. telophase: cleavage furrow forms, nuclear envelope reforms

centrioles

one is older than the other

duplication starts in s phase

prophase

disassembly of golgi, mito

dissociation of chromatids

mt dynamics v high

• XMAP inhibited, no stabilization

• only kinesin-13, which destabilizes mts

metaphase

breakdown of nuclear envelope through lamin phosphorylation

both kinetochores attached, chromosome moves toward spindle

settle at plate

kinetochore

aurora b kinase phosphorylates Ndc80 → loosens mt/kinetochore attachment

must be captured by mt on both sides to prevent aurora b from loosening attachment

• thus allows stabilization of interaction

anaphase

separation of sister chromatids

• typically held by cohesins

• separase can cut cohesins

  • must have securins ubiquinated and degraded before activation of separase

  • mediated by apc/c-cdc30

sac is emitted when kinetochores aren’t properly attached, inhibits apc/c-cdc20

• thus prevents separase from cutting adhesins, preventing anaphase entry

telophase

nuclear envelope reforms

cytokinesis

cleavage to produce two daughter cells

contractile ring of actin and myosin forms to divide the cytoplasm in two

use ATP to produce contractile force

requires vesicles to maintain membrane homeostasis

contractile ring location mediated by Rho GTPase

cytokinesis midbody

contains molecules that stimulate Rho GTPase to form a contractile ring

recruits GEF

Rho promotes actin formation and myosin

apoptosis

regulated cell death

cell shrinks/bubbles, loses ecm attachment

chromatin condensation → nuclear envelope broken

DNA fragmentation → mediated by endonucleases

ps exposure in outer leaflet → atp dependent flippase usually flips ps into innter leaflet, gets inhibited during apoptosis so ps is always in outer leaflet

dead cells get engulfed

necrosis

cell death caused by trauma or physical damage

cell swells

no chromatin condensation or dna laddering

inflammatory response → immune cells must neutralize the proteins that leak out

apoptosis pathway

intracellular proteolytic cascade mediated by caspases → proteases with cysteine

• synthesized as a pro-enzyme, must be activated after cleavage by other activated caspases

two types: intrinsic and extrinsic

bcl2/intrinsic apoptosis pathway

mitochondria

triggered by cell damage of stress

cytochrome c is normally stored within the inner mito membrane

• regulated by bcl2 proteins

• anti-apoptotic prevents release

• pro-apoptotic promotes release

  • these two can bind and inhibit eo

once released, it binds to Apaf1

activation causes change, leading to multimer formation and binding site exposure

procaspase 9 then binds to form apoptosome

activated caspase9 then activates the caspase cascade

bcl2 specifics

anti-apoptotic proteins prevent aggregation of pro-apoptotic proteins (bax, bak) in outer mito membrane

if apoptotic signal is received, pro-apoptotic proteins from abother faily inhibit anti-apoptotic blc2 proteins

fas/extrinsic apoptosis pathway

receptor on plasma membrane → Fas death receptor

trimeric Fas ligand binds to receptor and causes trimerization within membrane

conformational change recruits FADD protein and procaspase8

• procaspase8 cross-cleaves/activates eo

activated caspase8 can then activate the caspase cascade

caspase and dna fragmentation

caspase mediated activation of dna endonucleases lead to the fragmentation observed during apoptosis

apoptosis survival factors

stimulate anti-apoptotic proteins

inhibition of pro-apoptotic bh3 proteins by activation of protein kinases

require constant signals to promote cell survival

• cells only survive when and where needed

• bc cells compete for resources and stuff