MBM Block 3

Cholera Toxin → Cell Signaling Dysfunction =

Prevents GTP hydrolysis → Gαs remains active → ↑ AC activity → ↑ cAMP → ↑ fluid loss

H. pylori → Cell Signaling Dysfunction =

Peptic ulcers via excessive acid production

General Signaling Cascade Steps

1. Ligand binds receptor

2. Receptor changes in response to ligand

3. Intracellular cascade

4. Cellular response

Lipid-Soluble Ligand Characteristics

• Hydrophobic

• Intracellular receptor binding

• Direct interaction for response

• Ex. steroid hormones, thyroid hormones, NO

Lipid-Insoluble Ligands Characteristics

• Hydrophilic

• Extracellular receptor binding

• Indirect interaction for response

• Ex. peptide hormones, growth factors, NT

Step 1 of Lipid Soluble Direct Enzyme Activation

Blood vessels release NO

Step 2 of Lipid Soluble Direct Enzyme Activation

NO diffuses to smooth muscles

Step 3 of Lipid Soluble Direct Enzyme Activation

NO activates guanylyl cyclase → converts GTP to cGMP → cGMP activates PKG → ⬇ intracellular Ca2+

Step 4 of Lipid Soluble Direct Enzyme Activation

Smooth muscle relaxation → vasodilation

PDE =

Enzyme that breaks down cyclic nucleotides

• Allows smooth muscle relaxation response to eventually end

Viagra =

PDE inhibitor

• ⬇ cGMP breakdown

Nitrates → ___ cGMP

↑ cGMP production

Caffeine =

PDE Inhibitor

Step 1 Gs Pathway

Ligand binds GPCR on cell surface

Step 2 Gs Pathway

GPCR activates Gs alpha subunit

GDP → GTP

Step 3 Gs Pathway

Gas activates adenylyl cyclase → AC converts ATP to cAMP → cAMP activates PKA → PKA phosphorylates target protein

Step 4 Gs Pathway

Cellular Response

Gi Pathway

1. Inhibitory hormone binds receptor

2. GPCR activates Gi alpha subunit

3. Gi inhibits adenylyl cyclase → ↓ cAMP → ↓ PKA activity

4. No downstream effects

How to Stop G Protein Signal

• Unbind ligand

• Inactivate G protein via Inherent GTPase Hydrolysis

• Inactivate 2nd messenger via cAMP phosphodiesterase

• Dephosphorylate proteins via phosphatases

Step 1 of Gq Pathway

Ligand binds GPCR on cell surface

Step 2 of Gq Pathway

GPCR activates Gq alpha subunit

Step 3 of Gq Pathway

Gq activates PLC → splits PIP2 into IP3 + DAG →

• IP3 releases Ca2+ from SR → activates PKC

• DAG activates PKC

Step 4 of Gq Pathway

Cellular response → muscle contraction

cAMP pathway receptor =

B-adrenergic

Vasodilation

PLC pathway receptor =

a1-adrenergic receptor

Vasoconstriction

Step 1 of RTK-MAPK Pathway

GF binds RTK

Step 2 of RTK-MAPK Pathway

RTKs dimerize → autophosphorylate tyrosine residues

Step 3 of RTK-MAPK Pathway

Adaptor proteins bind to phosphotyrosines → GEF converts GDP to GTP → Ras activated → Raf → MEK → ERK cascade

Step 4 of RTK-MAPK Pathway

TF Phosphorylated → changes in gene expression, cell growth, proliferation, differentiation

Step 1 of RTK-PLC Pathway

GF binds RTK

Step 2 of RTK-PLC Pathway

RTK dimerizes → autophosphorylates tyrosine

Step 3 of RTK-PLC Pathway

Activated RTK activates PLCγ → PIP2 splits into IP3 + DAG

• IP3 → Ca2+ release

• DAG activates PKC

Step 4 of RTK-PLC Pathway

PKC causes cellular response

RTK vs Non-RTK

RTK: intrinsic kinase activity

Non-RTK: separate intracellular tyrosine kinase enzyme associated w/ receptor

Step 0 of Non-RTK Signaling

Separate tyrosine kinase associates w/ receptor

Step 1 of JAK/STAT Pathway

Cytokine binds non-RTK

Step 2 of JAK/STAT Pathway

Receptor dimerizes → cross phosphorylation

Step 3 of JAK/STAT Pathway

JAK activated → phosphorylates receptor → becomes a docking site for STAT → JAK phosphorylates STAT → STAT dimerize → STAT translocated to nucleus

Step 4 of JAK/STAT Pathway

Initiated transcription of target gene → immune responses, inflammation

Hedgehog Characteristics

Embryonic development, cell growth, and differentiation

Regulate gene expression through Gli

Hedgehog Step 1

Hedgehod binds Patched receptor

Hedgehog Step 2

Hedgehog prevents Patched from inhibiting Smoothened

Hedgehog Step 3

Smoothened activates Gli → Gli enters nucleus

Hedgehog Step 4

Gli inside nucleus activates transcription of target genes

NO Hedgehog =

Pathway OFF

• Patched inhibits Smoothened

• Gli not activated

• Target gene transcription OFF

Notch Characteristics

Nervous system development

Regulates expression through proteolytic cleavage

Notch Pathway Step 1

Delta binds Notch receptor on adjacent cell

Notch Pathway Step 2

ADAM cleaves Notch → cuts extracellular portion

Notch Pathway Step 3

γ-secretase cleaves Notch intracellular domain→ NICD translocated to nucleus

Notch Pathway Step 4

Activates gene transcription of Hes family

Abnormal JNK1 = Compromised MAPK signaling →

Neurogenerative disease (Alzheimer’s, Parkinson’s, ALS)

BCR-ABL →

• Abnormally active tyrosine kinase

• Causes uncontrolled cell proliferation

Gleevec (Imantinib) Drug Mechanism

1. Imantinib binds ATP-binding site of BCR-ABL

2. Prevents BCR-ABL from phosphorylating downstream proteins

3. ↓ Growth signaling → ↓ cancer cell proliferation

Drug Choice for Chronic Myeloid Leukemia or Acute Lymphoblastic Leukemia

Gleevec (Imatinib)

Tofacitinib Drug Mechanism

JAK Inhibitor

1. Competes with ATP for JAK kinase domain

2. Inhibits STAT activation + transcription

3. Decrease immune response

4. Blocks gut inflammation

Drug Choice for Ulcerative Colitis

Tofacitinib

HER2 →

• Overexpressed in cancer cells

• Promotes downstream growth signaling

Trastuzumab (Herceptin) Drug Mechanism

1. Binds to HER2 receptors on cancer cells

2. Reduces HER2-mediated growth signaling

3. Antibody recruits immune cells via Fc receptor

4. Immune cells attack HER2-expressing cancer cells → ↓ tumor cell proliferation

G1 Phase =

Growth

Most receptive to external signals

Can go into G0 phase → metabolically inactive

S Phase =

Synthesis

DNA Replication → Sister Chromatids

G2 Phase =

Continued growth

Synthesize proteins needed for mitosis/cytokinesis

M Phase =

1. Prophase/Prometaphase

   1. Dissolve nuclear membrane & chromosomes condense

2. Metaphase

   1. Chromosomes attach to spindles at kinetochores

3. Anaphase

   1. Separate sister chromatids → opposite poles

4. Telophase

   1. Nuclear membrane reformed

   2. Chromosomes decondense

Cytokinesis

Cell Division via Cleavage Furrow

Cleavage Furrow =

Contractile ring → actin filament create fissure → separation

CDKs

Ser/Thr kinases

• Phosphorylate target proteins

• Present throughout all cycle at equal levels but only active during certain phases

Cyclin

Bind/activates CDK

• Concentration increases when needed to activate specific CDK

G1-CDK = Cyclin ___

Cyclin D

G1/S-CDK = Cyclin ___

Cyclin E

S-CDK = Cyclin ___

Cyclin A

G2-CDK = Cyclin ___

Cyclin A (& B)

M-CDK = Cyclin ___

Cyclin B

CDK Cell Cycle Regulation

1. CAK binds/activates CDK

2. Cyclin binds/activates CDK

3. CDK activates target protein

4. Cyclin is destroyed

CKI: p27

Pries CDK and Cyclin apart

Present until GF is present

CKI: p21

Present in response to DNA damage

Prevents division of damaged DNA

Poluubiquination on Cyclin

Marks for proteosomal degradation

Proteasome →

Ubiquitin interacts with proteasome cap

Target protein → transferred inside proteasome → degraded

G1 → S Restriction Step 1

EGF signals Ras-MAPK → signals transcription of Cyclin D → activates G1-CDK (+CAK) → removed p27 → CDK phosphorylates target proteins (Rb)

G1 → S Restriction Step 2

Rb protein → releases E2F → allows transcription of Cyclin E

G1 → S Restriction Step 3

p27 inhibits Cyclin E / G1/S-CDK complex

At Restriction Point →

↑ Cyclin E synthesis

↓ p27 synthesis

Progress to S phase → no more external signals needed

G2 → M Progression

Cyclin B binds M-CDK → phosphorylates Lamin → helps dissolve nuclear envelope

G2 → M: Wee1

Phosphorylates Cyclin B/M-CDK → inactivates P of target genes → stockpile complexes until M phase

How does the Cyclin B/M-CDK complex get reactivated after Wee1?

Cdc25 removes Wee1 from complex

M → G1 Progression

APC targets Cyclin B for ubiquination → cyclin is degraded → cell cycle restarts to G1 awaiting GF

G1 Checkpoint

Check for GF and DNA damage

G2 Checkpoint

Check for complete DNA replication + DNA damage

Metaphase-Anaphase Checkpoint

Check chromosomal attachment to spindles

• Causes nondisjunction if bypassed

Paths of Detected DNA Damage

• Repair DNA or

• Arrest Cell Cycle

  • Apoptosis or DNA Repair

Growth Inhibitors

p53 + Rb

Tumor suppressors

DNA Damage and p53 Pathway

DNA Damage → ↑ p53 → Cell Cycle Arrest → DNA Repair OR Irreversible Damage → Apoptosis

p53 → p21

p53 increases p21 synthesis → p21 inhibits cyclin/CDK complex → cell cycle pauses → allows time for DNA repair

Irreversible damage → p53 ↑ transcription of apoptosis genes

M Checkpoint Step 1

Ensure spindle fibers are properly attached to kinetochores before anaphase

Cdc20 activates APC/C → proceeds to anaphase

What occurs if spindle fibers are not attached at M Checkpoint?

Cdc20 is inhibited

APC/C remains inactive

Cannot proceed to anaphase

M Checkpoint Step 2

Separate sister chromatids

Cohesin

Holds sister chromatids together

Securin

Bind/inhibit separase

Separase

Cleave cohesin → separate sisters

Metaphase → Anaphase Steps

1. Cdc20 activates APC/C

2. APC/C ubiquinates securin → securin degrades

3. Separase becomes active

4. Separase cleaves cohesion → sisters separate

5. Microtubules shorten → chromatids move to opposite poles

Spontaneous Cancers Progression

Chemoradiation →

Cause DNA damage → trigger p53 → apoptosis of cancer cells