Lecture #9 - Signling to the Nucleus

Question 1

Three ways that signaling to the nucleus happens

Answer 1

1. Immediate Cell-Cell interactions
2. Relay modalities
3. Long range signaling modalities (Ex. Steroid hormone signaling)

The receptor itself can be the transcrtion factor OR can have actaivtion pathways that lead to the translocation of the transcrtion factor to the nucleus

Overall signaling to the nucleus - Signal from outside of the cell is transduces into the nucleus to affect gene regulation

Question 2

Steroid hormone signaling - Overall

Answer 2

Four classes of steroid hormone receptors
- Most are class 1 –> bind ligand in cytoplasm and bind to DNA as a homodimer

Ligands of sterorid hormone receptors move freely through the plamsa membrane and NE

Steroid hormone pathway = long range communication/signaling (systemic signaling)
- Signal (ligand) moved through the blood to tissues

Steroid hormone signaling Receptor themeslves are TF

Steroid hormone signaling results in long lasting effect

Example Steroid Receptors - gluccotocoid + estrogen + androgen + progestrone

Question 3

Steroid Receptor Before and After ligand binding

Answer 3

Before ligand binding – steroid hormone receptor is sequestered in the cytoplasm by chaparones that prevent unfoldoing/agregating/dimerization of the receptor (Ex. Heat shock proteins)

Ligand (hormone) crosses plasma membrane and binds to receptor in cytoplasm –> ligand binding domain of the receptor undergoes confirmation chnages that allows the heat shock proteins to be removed and the free receptor/ligand complex translocates to the nucelus –> receptors dimerize in the nucleus

Question 4

Structure of the steroid hormone receptor family

Answer 4

Steroid hormone receptors share a modular architecture of independent functional domains

Conserved functional domains:
1. DNA binding domain (most conserved)
2. Ligand binding domain (C terminus) - contains the activation domain
- Need activation domain and DNA binding domain in order to have transcriptional activation
3. Flexible hinge region - separates the ligand bidning and the DNA binding domains

Question 5

Steroid Hormone Receptors in the Nucleus

Answer 5

Once in the nucelus - Steroid hormone receptors dimerize and dimers bind to regulatory elements in target genes (bind to the Hormone responsive element)
- Hormone responsive elements are often in a specific gene in a specific target tissue
- Example – Andeorgen Receptor binds to the androgen receptor resposive elements in regulatory regions of Prostate specific antigen (PSA) gene

Receptors CAN actaivte genes without hormone responsive elements through interactions with other proteins - Hormone Receptors can piggy back on other proteins –> other protein will bind to their own response elements
- Example - Progesterone binds to STAT –> STAT binds to STAT binding domains –> affects transcription

Question 6

Steroid hormone receptor signaling - Process

Answer 6

Start – ligand traveling in the blood bound to carrier proteins ; receptors in the cytoplasm in a non-oligomeric form

1. Ligand (hormone) goes into the cell using passive diffusion (could be taken in by endocytosis of the carrier proteins)
2. Hormone binds to the receptor
3. Steroid hormone receptor is released from the chaprones
4. Steroid hormone receptor/ligand complex translocates to the nucelus
5. In the nucelus the steroid hormone receptor dimerizes and the dimer binds to the hormone responsive element
6. When dimer binds to the HRE – dimers remove co-surpessers and interact with co-regulators to affect transcription

Question 7

Studying Dynamic Cellular localization of receptors

Answer 7

Use - Study Subcellular localization/dymanics of steroid hormone receptors in living cells using fusion contracts with GFP

Show that steroid hormone receptors can be divided into 3 groups based on their ligand free state distrubution:
1. Found in the nucleus (Ex. Estrogen Receptor)
2. Found in the cytoplasm (Ex. AR)
3. Mixed distribution in the cytoplasm and the nucleus (Ex. PR)

Studies found – Rapid and complete translocation of the receoptors to the nucelus once the ligand is added + ALL steroid hormone receptors dimerize in the nucelus

Question 8

Experiment - steroid hormone receptor signaling

Answer 8

Experiment - Fused GFP to the androgen receptor (shows the localization/translocation of the AR)

Found:
No ligand bound - AR is in the cytoplasm waiting for the ligand to cross the membrane

Add andogogen ligand – GFP signal goes to the nucelus –> AR goes to the nucelus
- GFP signal in the nucelus has a non-unifrom distrubution (in patches) = andorgen receptor has non-uniform distrbution

Question 9

Dimerization of Steroid Hormone Receptor

Answer 9

Steroid Hormone Receptor typically form homodimer

Dimerization is facilitated through interactions between the LBDs of both receptors
- Ligand binding domain binds to the ligand AND have an activation domain AND fosters the dimer formation

Dimers = affective form of receptor that allows for DNA binding and transcriptional changes

FRET studies on AR – Shows dimerization takes place after ligand binds AND happens in the nucleus

Question 10

FRET

Answer 10

Use - Detects dimerization + confirmational changes (shows when and where both occur)

Use #1 – express 2 versions of protein (1 is CFP taged and 1 is YFP taged) –> ONLY when the taged proteins are close can the signal transfer and get florunece of YPF (shows have a homodimer)
- Only get YFP in the nucleus –> only get dimerization in the nucleus

Use #2 – Can see confimation changes within 1 protein - Add CFP and YFP at diferent places on the protein –> TEHN add ligand –> See when there is YFP signal to see when the confirmation change happens

Question 11

Regulation of steroid hormone receptor pathways and interactions with other pathways

Answer 11

Steroid hormone receptors are subject to many phosphorylation events to regulate their functions (phosphorylation = regulation sites ; affects activity of receptor)
- Because of use of phosphorylation for regulation - Steroid hormone receptors inetarct with/ are modulated by other signaling pathways in cell (ras/MAPK kinase pathway can affect phosphorylation of receptor)
- Pathway is modulated by all other signaling pathways that are happening in the cell

Question 12

Activation of steroid hormone receptors without ligand

Answer 12

Example – Estrogen receptors are always in the nucleus

Once ligand binds to the receptor –> estrigen receptor dimerizes in the nucleus –> Dimer binds to the hormone response elements (ER is the TF)
- ER is also regulated by phosphorylation
- Because the ER is always in the nucelus when there is no esterdiol (no ligand) - the ER can go onto chromatin at low levels to affect transcription

Question 13

Cross talk between pathways

Answer 13

Cross talk between pathways it important – pathways is regulated by other signaling pathways AND the outcome of a pathway is affected by other signaling pathways happening in the cell

Question 14

Type of Steroid hormone Receptor - Class 2 Nuclear receptors

Answer 14

Class 2 nucelar recetors - bound to DNA as a heterodimer in the nucleus BEFORE the ligand binds

No ligand – Receptors recruit co-repressors (Repress target gene)

With ligand –> Receptor has a confirmation chnage –> Co-repressors leave –> Receptor recruits co-activators complex –> activate the target genes

Example - Thyroid hormone receptors

Question 15

Regulation of the Stroid receptor Action

Answer 15

1. Pathway depends on nuclear import and export signals –> use nuclear import/export as a form of regulation (Ex. increase nuclear export to modulate strength of signaling)
   - Use NLS/NES or importins/exportins for regulation of receptors
   - Hsp proteins can block NLS and NES when receptor is in the cytosol
2. Proteosome degrades receptors over time (pathway is self deliniating)
3. Interaction with co-actaivtors/what co-actiavtors are in the cell + how protein interacts with the DNA affects which genes the steroid hormone receptor turn on/off
   
   • Example – receptor can bind to the HRE OR can interact with other proteins –> lead to activation of different genes

Question 16

Affect of nuclear receptors on transcription

Answer 16

Hormone Receptors can activate or repress gene transcription

Example – Class 2 receptors
- No ligand - bind to the HRE and recruit repressor complexes –> repress transcription of the target genes
- Once ligand comes in –> activate genes

Question 17

Notch Signaling - Overall

Answer 17

Direct cell-cell communication (Cells are in direct contact)
- Cell-cell –> Ligands on the plasma membrane of one cell AND the Notch receptor on the plasma membrane of the second cell –> have change in gene expression in the signal receiving cell (cell bound to Notch)

In Notch - the receptor is the transcription factor

Pathway is important in cellular development and tissue homeostasis (cell proliferation + differentiation + apoptosis)

Notch signaling pathway has 2 roles depending on how they are organized:
1. Tissue growth/cancer
2. Cell death/tumor suppression

Question 18

Ligands for Notch signaling

Answer 18

Ligands that bind to Notch Receptors:
1. Jaggard Family
2. Delta Family

Ligands are in the cell surface of the adjacent cell (have 1 transmembrane domain)

Delta and Jaggard are glycoproteins

Ligand bound cell = sender (transmitting) ; Notch bound cell = receiver

Question 19

Structure of Notch Receptor

Answer 19

Notch receptor is composed of two non-covaltley linked components:
1. Extracelular Subunit
2. Integral glycoprotein

Newly transcribed Notch protein undergoes Furin cleavge (S1 Cleavage) and fringe glycosylation in golgi–> ONLY after cleavge and glysylation the Notch protein gets put back together non-covalentley –> non-covatley attatched protein goes to the cell surface

Question 20

Notch Pathway

Answer 20

1. The ligand (delta) binds to Notch –> binding leads to the activation of a proteolytic complex in the transmembrane glycoproteins (leads to S3-Cleavage)
2. Proteacitic activity (gamma secretase) cleaves Notch at cleavage site 3 to make the Notch inracellular domain (NICD
3. Released NICD goes into the nucleus
4. In the nucleus NCID binds to CSL –> binding removes the co-repressors and recruits co-activators (MamL and p300) –> enable the activation of the target genes
   - Before Notch goes to nucelus - CSL in a complex with co-repressed on the DNA
5. When NCID is phosphorylated in the N-terminal PEST domain Inactivation of target genes transcrtion begin
6. After phosphorylation – NCID will be ubiquinated and go to the proteosome for degradation

Question 21

Effect of ligand binding to Notch receptor

Answer 21

Ligand binding leads to the activation of a proteolytic complex (Ex. Presenlin) –> Proteolytic complex completes S3-Cleavage

Activation of proteolyic cleavage - Once ligand (delta/jagged) binds to receptor –> Delta/Jagged are endocyted –> During endocyrosis Delta/Jaggad ligands pull on notch –> exposes Cleavage Site (S2) –> NOW (after endocytosis) ADAM/gamma secretase proteolytic activity can cleave to release inrecalular region (NCID) into the Notch bound cell
- Extracellular region of Notch is endocytsoed by the ligand expressing cells (sending cells) via a E3 ligase (mindbomb)

Question 22

Turning off Notch Pathway

Answer 22

NEED to be able to turn genes off (deactivation of target genes)

Inactivation of target genes transcrtion is done by phosphorylating NCID in the N-terminal PEST domain
- Phosphorylation is done by Cyclin dependnt kinase 8

In lab – used to get more protein turnover of proteins that are expressed in cells (PEST domain = important for turnover)

Question 23

Regulatory points in Notch signaling

Answer 23

1. Regulation of cleavage of Notch Protein
2. Regulation of the Interaction of Delta/Jagged with the Notch Receptor

Question 24

Live cell techniques

Answer 24

Use - Determine the role of and importance pathway proteins on Notch localization/functions (see important components of pathway)

Experiment – Fuse Fragment of Notch protein to GFP

Results:
- If block gamma secretease activity (Notch is not cleaved) –> Notch can’t go to the nucelus (stays in the cytoplasm or the plasma membrane)

Question 25

Do all cells have the same response when Notch is activated

Answer 25

Different cells can have the same signaling moelcules that go into the nucleus BUT can get a different outcome depending on the cell type The response is affceted by: 1. The co-actaivtors in the cell 2. Other signaling pathways in the cell that are competing with the Notch 3. The chromatin state in the cell (chromatin state is different in different cells) - Chromatin state affects the response to signaling

Question 26

Regulation of Notch-Meidated Transcription

Answer 26

Regulate using chromatin state In the Notch target genes (have CSL bound to them) --> CSL and BRM are on poised genes – chromatin is not closed but it is not open - Poised genes have some nucleosome turnover Have a poised locus so that when NCID goes into the nucleus --> NCID interacts with CSL --> interaction causes higher nucleosome turnover --> NCID is able to activate the gene - Posing of the target genes is important for notch to be able to activate target genes (need chromatin a little open) Different cells response to signaling (Ex. Notch) differently because they have different chromatin states (Ex. NCID going into a cell with posied chromatin has a different outcome than NCID going into a cell that does not have poised chromatin)

Question 27

NfkB signlaing - Overall

Answer 27

Relay Signaling Pathway - NfkB is downstream of other signaling pathways In NfkB the receptor is NOT the transcription factor (NfKB can regulate transcription factors) NfkB reponse to multiple signaling pathways (involved in cell response to stress, cytokines, free radicals, bacterial and viral antigens) - NfkB is important in B cells and immune function (adaptive and innate immune response) NfkB is found in almost all animal cell types

Question 28

NF- κB family of transcription factors

Answer 28

NF- κB family of transcription factors includes five subunits: 1. p50 2. p52 3. p65 (RelA) 4. c-Rel 5. RelB Subunits can homo- and heterodimerize Having different SU is a form of regulation - get different signals that come in depending on what SU dimerize

Question 28

IkB

Answer 28

IkB = inhibitor of NF-kB (Ikb regulates localization of Nfkb) - Nf-Kb action is linked to the activity of IkBs - IkBs proteins includes – Ikba, Ikb-beta, Ikb-gamma, Ikb-epsilon, BCL3 Ikb is the TARGET of upstream signaling cascades Phosphorylation of Ikb leads to proteosomal degredation of IkB Structure of IkB - Do NOT have Transactivation domain ; Have Ankyrin repeats that mediates the assmebly of IkB with other Rel proteins

Question 29

Structure of NF-kB and IkB proteins

Answer 29

N-terminus - ALL NF-kB proteins have a Rel homology domain C-terminus - SOME Nf-kB proteins have a transactivating domain that is able to activate transcription (ONLY found in RelA, RelB, c-Rel) Class 1 Nf-Kb and IkB proteins are synthesized as larger precursors (p105 and p100 are the larger precursors of p50 and p52 subunits) - p100 and p105/p50 and p52 do not have TAD (bind to DNA but can't actually do anything)

Question 30

IkB bound NfkB

Answer 30

IkB is bound to Nf-kB Rel proteins --> Rel NLS signal is masked (can't go to nucelus) - NfKb proteins uses Nucelus localizatiion as a point of regulation/control Removal of IkB - Done by phosphorylation of 2 serine residues (SS) at the N temrinus of IkB --> phosphorylation triggers polyubiquination and proteosome-mediated degradtion of IkB

Question 31

NfkB and IkB signaling pathway

Answer 31

Start (inactivated) – NfKb heterodimer of Rel (has TAD ; can activate DNA) and p50 is formed ; IkB is bound to the dimer so the IkB/dimer complex stays in the cytoplasm 1. Extracellular signals (liagnds) bind to membrane receptors --> activates IkB Kinase --> IkB kinase phosphorylates IkB 2. Phosphorylation of IkB signals for IkB to be ubiquinted/degraded in proteosome and for its dissociation from NfkB (NOW the Rel-p50 NfKb heterodimer is free) 3. Activated Nf-Kb is translocated to the nuelus --> NfKb dimer binds to repsonse elements in DNA --> binding of Nf-kb recruits Co-actiavtors and recruits RNA polymerase - NfkB dimer = TF (affects transcription)

Question 32

Dimers made in Nf-kB signaling

Answer 32

Three types of dimers determine downstream gene regulation: 1. P50-p50 --> sits on DNA and prevents a activating NfKb dimer from binding - Dimer can't activate transcription because neither have TAD - P50-p50 dimer = repressors 2. C-Rel and P50 ; RelA-p52 ; RelB-p52 or p50 --> have 1 TAD --> recruits co-activators and affect transcription 3. RelB-p52 or p50 --> have 1 TAD --> can recruit co-activators and affect transcription Combination that makes of heterodimer affects transcription or repression of downstream genes

Question 33

Example NfKb pathways

Answer 33

Example - Tumor necrosis factor Pathway 1. TNF binds to the TNF receptor --> causes receptor olgiomerization --> oligiomerization recurits adapters TRADD and TNF receptor associated factor (TRAF proteins - E3 ub ligase) - Ubiquination of Rip1 and Traf2 are important for scaffolding 2. Additional proteins scaffold in a ubiquitin dependent manner – After Scaffolding the receptor complex traps Tak 1 (GF activated kinase) 3. Tak 1 kinase phosphorylates Ikb Kinase --> IkB kinase dissociates from NF-Kb essential modifier (NEMO) --> Ikb Kinase phosphorylates Ikb --> IkB is polyubiquinated and degraded by the proteosome --> NfkB p50-p65 heterodimer is released 4. NfKb p50-p65 heterodimer is imported into the nucelus --> Nf-kB binds to NfkB promoters elements and activates expression of many genes

Question 34

Scafolding in NfkB signaling

Answer 34

Scafolding is important in NfkB signaling because scaffolding signals for and results in the phosphorylatoon of IkB kinase

Question 35

How does expression stop in Nf-kB pathway

Answer 35

Transcription stops when NfKb is exported from the nucelus To help turn off pathway - Target genes of p50-p65 will make Ikb (Need to make IkB because it is degraded after phosphorylation) - Because make IkB - once NfKB is exported it will bind to new IkB (inhibited) and signaling pathway stops -NOW Nf-kB won't just go back into the nucelus after it is exported (makes sure there is no futile cycle) - Making IkB makes the pathway self deliniating (can eventually stop pathway) - Shows nuclear import/export are important

Question 36

Role of NfkB pathways

Answer 36

NFkb regulates development and immunity in flies and humans In development - uses the dorsal pathway (uses NfkB in a different context) - Fly Dorsal pathway and human NfKb pathways are similar

Question 37

How is the NfkB signal interpreted by the genome

Answer 37

Way signal is interpreted by genome: 1. Different types of heterodimers that can bind to DNA and be interprted by cell and lead to transcription of specific genes 2. Encoding 3. Decoding ALL would give a different outcome depending on other cellular and extracellular siganls Encoding and Decoding show Pathways are affected by factors and what is happening inside of cell (get different transcription in different cell types because of encoding and decoding/because of other things found in cell) I THINK - ALL shows form of regultion

Question 38

Way signal is interpreted by genome - Encoding

Answer 38

Encoding – Things you can increase/decrease to get proteins to interact with DNA in a certain way (things going into nucelus) Overall - Stimuli type and duration affect how the genomic outcome of the signal (leads to different gene programs) 1. Temperal chnages in stimuli (doses) -IF always have a lot of signlaing --> proteins keep going to the nucleus --> get increase in siganl Vs. intermediate amount of signlaing --> have different interaction of proteins going to the nucleus 2. Stimuli type

Question 39

Way signal is interpreted by genome - Decoding

Answer 39

Decoding (changing things within the nucelus) – Nf-kB goes to the nucelus and interacts with factors to read out different potential programs (how are things read by the cell) 1. Chromatin state affects how things are read by cell (affects transcriptional change) - Can have regions of the genome have open chromatin --> more responsive when Nf-Kb binds Vs. Regions that are heterochromatinized would need more/stronger signals to get transcription of genes 2. Decoding is affected by the level of the inhibitor (affects how signal affects genome) 3. mRNA stability and nucelar export/import

Question 40

Wnt Signaling - Overall

Answer 40

Wnt (signaling molecule/ligand) = growth stimulatory factors - Wnt receptor is a GPCR Pathway uses Beta catenin (effector molecule that acts in receiving cell) - Beta catenin = Regulated by degradation - Beta Catenin = Transcription factor Wnt pathway = has a role in embryonic development (development of heart) + affects carcinogensis There are many downstream events that result from Wnt Signaling (Ex. changes in gene expression)

Question 41

Wnt Pathway - NO Wnt signaling

Answer 41

Beta Catenin is continously made in the cell --> Beta catenin is brought into/is part of Beta catenin degradation complex - Degredation complex includes Axin (scafolidng proteins) + APC + pP2A (phosphatase) + CKIa (Casein Kinase) + GSK-3b (Glycogen synthase kinase) + beta catenin Within the complex – CKIa phosphorylates beta catenin (primes beta catenin) --> GSK-3b phosphorylates beta catenin --> phosphorylated beta catenin is recognized by F-box/Beta-TrcP/ubiquitin ligase complex --> beta catenin is ubiquinated and targeted beta catenin for destruction by the proteosome NOW there is less beta catenin in the cells

Question 42

Transcription of genes in Wnt pathway when Beta Catenin is degraded

Answer 42

Beta catenin is deraded = there is less beta catenin in the cells --> NOW there is no beta catenin to bind to target genes --> the target genes are repressed

Question 43

Wnt Pathway when have Wnt

Answer 43

1. Wnt (ligand) brings together the cysteine rich domain of frizzled receptor (GPCR) and the frizzled co-receptor (LRP5/6) (BOTH surface proteins) 2. Membrane bound CKI phosphorylates LRP5/6 co-receptor --> Axin (scafolding proteins) binds to the phosphrylated regions of LRP5/6 co-receptor --> Dishevled binds 3. When have the Axin/Dishevleved complex at the plasma membrane --> GSK-3b is inhibited --> newly synethsized beta catenin is not degraded --> beta catenin accumulates in the cytoplasm and can go to the nucleus and activate transcription - Uses mass action (have lots of beta catenin so now can go to the nucleus ; before it was degraded)

Question 44

Pathway beta Catenin Interactions

Answer 44

Beta-catenin has multiple nuclear binding partners (interacts with many transcrtional activators including cell type specific and general transcription factors) - MEANS pathway results in cell type specific and global outcomes Example – have a context dependent factor that is involved in enhancer looping with Wnt response elements (WRE) - Beta catenin helps build of complexes on WRE - In complex - Have many docking sites for cell type sepcifc Transcripton factors

Question 45

Sonic Hedgehog Pathway - Overall

Answer 45

3 hedgehog genes in humans - desert + indian + sonic hedgehog Hedgehog = ligand (Binds to receptor)

Question 46

How is Hedgehog protein made/secreted

Answer 46

Hedgehog – made as a larger protein --> Choletral groups group is added to the N terminus of hedghog protein by the C terminus of hedghog --> hedgehog protein is cleaved --> the C terminus goes to the proteosome while the rest of the protein (N terminus with the cholestral) go to teh ER and secretroy patwhay where skinny hedghog adds palmitic acid to the N terminus END – hedgehog protein = sticky protein (Will stick to any membrane)

Question 47

Ways for hedghog to leave the secretory pathway (ways to leave cell and affect signaling of nearby cells)

Answer 47

Ways to leave: 1. N terminus of hedghog interacts with SCUBE2 proteins --> hedgehog is released from plasma membrane 2. Hedghod ligand multimerizes --> Secreted hedghog as a sticky multimer complex 3. Hedghog interacts with heprin and glycocan porteins --> makes vescile --> Secreted hesdhoge as a sticky complex 4. Hedhgod enters a exovesicle --> Secreted hesdhoge as a sticky complex IN ALL - making a stikcly molecule that will stick to membrane it bumps into

Question 48

Hedgehog signaling in use pathways

Answer 48

Hedgehog signaling controls cell patterning and differentiation (involved in embryonic development) - Hedgehog signaling has roles in organ/tissue specifc gene induction - Shows hedgehog is a local actor Because it is a sticky protein it will stick close by and make a gradient of protein depending on how much of the protein is secreted - Will have more of the hedgehog protein close by the site of secretion and a gradient that forms (less protein as go farther out)

Question 49

Type of signlaing in hedghog

Answer 49

Hedgehog signaling has graded signaling - Gradient of hedgehog protein induces different subsets of genes Gene activation in response to hedgehog occurs at different thresholds (gene activation is different depending on how much signaling there is) - Different levels of hedgehog = get different levels of signaling Response is based on Ratio of ciR/ciA (GLI in vertabrets) - CiR = effector proteins (

Question 50

Hedgehog signaling in invertebrates - Inactivated state

Answer 50

In inactiavted state (NO Hedghog) – Patch protein (in membrane) blocks activity of SMO - Patch = receptor for hedgehog When patch blocks SMO --> Sufu is blcoking Fu AND Ci protein is phosphorylated by PKA and CKI and GSK3b Phophsorulation of Ci --> leads to Cir (get repressive form) --> CiR goes to the nucelus --> CiR represses target genes

Question 51

Hedgehog signaling in invertebrates - Activated state

Answer 51

Activated State - When hedghog binds to patch --> SMO goes to the plasma membrane AND patch is repressed --> SMO is not inhiibted --> PKA, CKI, GSK-3b will phosphorylate regions of SMO --> CiA stays CiA and goes to the nucleus and affects transcription

Question 52

Interaction of Hedghog with other pathways

Answer 52

Other signaling pathways can affect if hedgehog pathway is able to have actiovation or repressive effects on tareget genes BECAUSE Proteins that phosphorylate Ci are coming from other signaling pathways = whether of not Ci is phosphorylated is based on other pathways

Question 53

Hedgehog signaling in vertebrates - Inactive state

Answer 53

At rest patch (PTC) (hedgehog receptor) is in the membrane inhibits the activity of SMO - Inhibited SMO is in the cytoplasm ; - SMO = seven membrane spanning receptor When SMO is inhibited – SMO is not able to act on a complex of cytoplasmic factors (BT-RCP) --> BT-RCP is recrited to GLI proteins --> sends complex to the proteosomes where GLI3 is partially degraded --> get truncated version of GLI3 --> truncated version of GLI3 is the repressor form of hedghog --> enters the nucelus --> target genes are turned off - Complex of cytoplasmic factors = regulate GLI1/2/3 (transcription factor)

Question 54

Hedgehog signaling in vertebrates - Active state

Answer 54

Pathways is on: 1. Hedgehog binds to Patch (PTC) receptor --> PTC won’t inhibit SMO --> SMO is recruited to the plasma membrane and Patch is endocytsosis/goes to lysosome --> SMO acts on other scaffolding proteins at the plasma membrane 2. NOW have SMO at the plasma memnrane --> SMO as on other scafolding proteins 3. GI intreupts the interaction of GLI1/2/3 with the suprressor fused complex which releases GLI1/2 is released from its latent form --> GLI1/2 is translocated to the nucelus --> In nucleus GLI1/2 activates transcription of target genes - GI is associated with SMO - GLI1/2 = activator forms of GLI protein

Question 55

SMO in active Vs. inactive Hedghog pathway

Answer 55

When inactive --> SMO is inhibited it is in teh cytopalsm When Active --> SMO us not inhibited SMO goes to the plasma membrane and is a GPCR

Question 56

SHH-regulated gene expression profiles

Answer 56

Hedgehog is a sticky signaling molecule --> when cells secretes the sticky signlaing molecule it will land on/stick to the close adjacvent cells the most As make more of the hedgehog sticky signaling molecule --> spread the signal out END - regions that are closest to the signaling cells that are secreting hedgehog will get the most hedgehog vs. the cells that are far away get the least hedgehog - Where a cell is relative to where the signal is being released leads to different levels of hedgehog --> different levels of hedgehog will lead to activation of different genes programs

Question 57

Use of graded signlaing in hedghog

Answer 57

Used for patterning genes during development – thresholds are important in setting up patterning identity

Question 58

Form of regulation used in hedghog (applies to other pathways)

Answer 58

Can regulate target gene activation using subnuclear localization of GLI1 (regulation is affected by where the transcription factor is in the nucelus) Example – GLI1 TF can be regulated by sequestering it to the nucelar lamina or through interactions with nucleoplasm factors

Question 59

Overall regulation of signaling pathways

Answer 59

Regulation of signaling pathways: 1. Ligand/receptor interactions 2. Interactions with other signaling pathways 3. PTMs to effector molecules (Ex. Kinases in cells) 4. Protein protein interactions (Ex. Co-factors may or may not be cell) 5. Translocation into the the nucleus/export from the nucelus 6. Sub-nuclear localization 7. Limited lifetime/attenuation (Ex. Protein can be exported or degraded so the pathway is not perminatley activated) 8. Interaction with or alteration of chromatin (Ex. Is the chromatin open or closed ; how does the effector or thing that is being recruited affect chromatin)

Question 60

Cross talk between pathways

Answer 60

There is cross talk between pathways pathways are all interacting and can and do affect one another

Question 61

Use of nuclear export in regulation

Answer 61

Exprot allows for attenuating signal Pathways use Regulation by Nuclear import and export - Used for sequesteration and attenutaion of signal

Question 62

Use of nuclear localization for regulation

Answer 62

Nuclear lamina and nuclear localization --> help attenuate signaling because it sequesters proteins to the nuclear lamina Example – Wnt, TGF-b (Nf-kB), Notch, Map kinases, AKt/mTOR --> ALL pathways that are attenuated by proteins in the nuclear lamina

Question 63

Degredation as a form of regulation

Answer 63

Degredation helps attenutae the signal Example - 1. Want to get rid of Notch intracellular domain using the PEST domain 2. Make more of an inhibitor (makes more of the off signal) - In NfkB pathway making more IkB END – always need a way to turn off signaling

Question 64

Chromatin Landscape and signaling as a form of regulation

Answer 64

Regulate by having different chrmoatin states (closed vs. Permissive Vs. Open chromatin) Open is more responsive to signaling molecules Vs. Close chromatin is harder to overcome

Question 65

How signaling interacts with chromatin

Answer 65

There are many ways to modify other chromatin modifying enzymes Example – Transcription factor binding can chnage chromatin state OR other chromatin modifying enzymes that modify chromatin

Question 66

Exampe – Notch signaling and gene activation (Example of regulation)

Answer 66

Brahma Swi/SNF chromatin remodeling complexes are required to “poise” the enhancer for Notch binding - Complexes keep target genes poised --> NOW when the signal comes in the genes is turned on at high enough levels to affect cellular fate SHOWS that in the absence of signaling – there are other molecules that allow target genes to be more ready for activation

Question 67

Effect of Notch binding and turnover

Answer 67

Notch binding and turnover alter chromatin Notch is degraded by PEST domain AND also have rebuilding of repressor complex (both ways to turn pathway off) - Having off pathway - creates a cycle of repression and activation in the nucleus (Depending on how strong and constant signaling is the cycle might be faster or slower) - Genes are not usually all on or all off there is a mix where certain loci are in different stages in the cycle

Question 68

Summary

Answer 68

Question 69

General Themes for pathway regulation

Answer 69