Cell Survival and Growth Control Notes

Pathways of Cell Survival and Growth Control - Part 2

Integration of Multiple Signals

Cells integrate multiple signals to produce the required response. Key pathways involved include:

  • Ras-MAP kinase
  • PI 3-kinase-PKB/Akt
  • PTK receptor

These pathways influence various cellular processes:

  • Apoptosis
  • Cell division
  • Cell survival

Strong signals from these pathways working together are crucial for proper cellular function.

Ras-MAP Kinase Cascade

The Ras-MAP kinase cascade is a growth factor-activated signaling pathway employed by many protein-tyrosine kinase (PTK) receptors. This pathway leads to changes in:

  • Gene expression
  • Cell cycle progression

Cyclin/CDK Control Points in the Cell Cycle

The cell cycle is regulated by Cyclin/CDK complexes at various control points:

  • G₁ phase: Cyclin D/CDK4 and Cyclin D/CDK6 complexes are active.
  • S phase: Cyclin E/CDK2 and Cyclin A/CDK2 complexes are active.
  • G₂ phase: Cyclin A/CDK1 complex is active.
  • M phase (Mitosis): Cyclin B/CDK1 complex is active.

Regulation of MYC Expression by Ras-MAPK Pathway

The Ras-MAPK pathway regulates MYC expression:

  1. Mitogen activates a mitogen receptor.
  2. This leads to Ras activation.
  3. Ras activates the MAP kinase cascade.
  4. MAP kinase activates a gene regulatory protein.
  5. MYC gene expression is increased.
  6. Increased MYC leads to increased Cyclin D and SCF subunit gene expression, and E2F synthesis.
  7. Increased Cyclin D leads to G₁-Cdk activation (Cyclin D-Cdk4).
  8. G₁/S-Cdk activation (Cyclin E-Cdk2) occurs.
  9. Rb phosphorylation increases, leading to increased E2F activity and entry into the S phase.

MYC, p53, and E2F Effector Pathways

These pathways are interconnected and regulate various cellular processes:

  • MYC:
    • Cell-cycle progression (Cyclin D2)
    • Metabolism (CAD, SHMT2, ODC, LDH)
    • Ribosome biogenesis (Nucleolin, BN51)
    • DNA/nucleotide biogenesis (Cyclin E, Cyclin D1)
  • p53:
    • Apoptosis (PERP, NOXA, APAF1, PUMA, PIG3)
    • Cell-cycle arrest (WAF1, 14-3-3σ, E2F)
  • E2F:
    • Cell-cycle progression (BMYB, DHFR, Thymidine kinase, DNA polα, CDC6, PCNA)

Too Much MYC Protein

Excessive MYC production can lead to cell cycle arrest or apoptosis:

  1. Excessive MYC production.
  2. This activates P19ARF which stabilizes and activates p53.
  3. Active p53 causes cell-cycle arrest or apoptosis.
  4. Mdm2 is involved in the degradation of p53.

Growth Factors Regulate Cell Cycle

Growth factors regulate the cell cycle through Cyclin D kinases:

  • Mitogens activate CDK4/CDK6 via Cyclin D.
  • INK4A inhibits CDK4/CDK6.
  • RB (retinoblastoma protein) is phosphorylated by Cyclin D/CDK4/6.
  • Phosphorylated RB releases E2F, leading to transcription of S-phase genes.
  • E2F promotes G1-S phase transition.
  • Differentiation and apoptosis can also occur.

Cell Cycle Targets of Ras/Raf Signal Transduction Pathways

Ras/Raf signal transduction pathways target various cell cycle components:

  • Growth factor signals activate Ras, which activates Raf.
  • Raf affects Jun, Cyclin D, and cdc25.
  • Cyclin D activates cdk4.
  • Myc is activated, affecting Cyclin E.
  • Cyclin E activates cdk2.
  • E2F is activated and phosphorylates Rb.
  • Phosphorylated Rb leads to the S phase.

PI 3-Kinase–Protein Kinase B Signalling Survival

PI 3-kinase and Protein Kinase B (PKB/Akt) are central to cell survival signaling:

  • PI stands for Phosphatidylinositol, an inner leaflet plasma membrane phospholipid.

Survival Signals

Cells require constant survival signals:

  • Growth factors
  • Cell-cell and cell-matrix contacts via integrins

Roles of PI 3-Kinases

Activators: fMLP, Thrombin, Integrins, B and T cell activators, Cytokines, Insulin, Growth Factors, Ras, Calcium

Functions of PI 3-Kinase:

  • Oncogenic transformation
  • Differentiation
  • Protein trafficking
  • Cell survival
  • Regulation of cytoskeleton secretion
  • Insulin-stimulated glucose uptake
  • Oxidative burst
  • Cell migration chemotaxis

Problem: Much of above defined using inhibitors!

Inhibitors: Wortmannin and LY294002

PI 3-Kinases (PI3K) Classes

  • Class IA PI3K: activated by protein tyrosine kinases (p110α, β, δ plus p85α, β, γ)
  • Class IB PI3K: activated by GPCRs (p110γ/p101)
  • Class II PI3K
  • Class III PI3K

Distinct 3-Phosphoinositides

PI3Ks produce various 3-phosphoinositides:

  • PtdIns(3)P
  • PtdIns(3,4)P2
  • PtdIns(3,5)P2
  • PtdIns(3,4,5)P3

These are generated from:

  • PtdIns
  • PtdIns(4)P
  • PtdIns(5)P
  • PtdIns(4,5)P2

PI 3-Kinase Interactions

PI 3-kinase products interact with various proteins:

  • PtdIns-3-P interacts with Lyn, Rab5, EEA1, AP2
  • PtdIns-3,5-P2 interacts with ?
  • PtdIns-3,4-P2 interacts with Btk, PLC, Vav2, Grp1
  • PtdIns-3,4,5-P3 interacts with Akt, PDK1, PKC

These interactions affect:

  • Vesicle trafficking
  • Cell survival (PFK, BAD, GSK3)
  • Ca2+ regulation
  • Proliferation (p70S6-K)
  • Vesicle budding
  • Cell migration (Rac, Arf)

Lipid Binding Domains

Various lipid-binding domains exist:

  • PH domains
  • FYVE domains
  • PX domains
  • C2 domains

Inositol Lipid Specific Binding Domains

  • PH domain bound to Ins(1,3,4,5)P4
  • Tubby with PtdIns(4,5)P2
  • ENTH of Epsin
  • SH2 of p85α
  • PX of p40phox

Use of GFP Signalling Domain Fusion Protein Reporters

GFP (Green Fluorescent Protein) can be fused with lipid-binding domains to visualize signaling events.

Absence of Survival Factor

In the absence of a survival factor:

  • PI 3-kinase is inactive.
  • BAD is not phosphorylated and inhibits anti-apoptotic proteins.
  • PKB is inactive in the cytosol.

Survival Signal: Factor Added

When a survival factor is added:

  • Receptor tyrosine kinase is activated.
  • PI 3-kinase is activated.
  • PKB is phosphorylated and activated.
  • BAD is phosphorylated and inactivated, promoting cell survival.

Receptor Activation and Autophosphorylation

Receptors are activated and autophosphorylated on tyrosine residues upon ligand binding.

PI 3-Kinase Recruitment

PI 3-kinase is recruited to the receptor via SH2 domains.

PI 3-Kinase Interaction with Substrate

PI 3-kinase interacts with its substrate PI(4,5)P₂.

Phosphorylation by PI3K

PI(4,5)P₂ is phosphorylated by PI3K to form PI(3,4,5)P3.

Recruitment of PDK1 & PKB

PDK1 & PKB are recruited to membranes via PH domain interactions with PI(3,4,5)P3.

PDK1: 3-Phosphoinositide-Dependent Protein Kinase

PDK1 is a master kinase in insulin and growth factor signaling pathways.

  • Activates a number of AGC family protein kinases.

PDK1 Activates PKB

PDK1 activates PKB by serine/threonine phosphorylation.

Activated PKB Dissociates from PDK1

Activated PKB dissociates from PDK1 after phosphorylation.

PKB Phosphorylates Substrates

PKB phosphorylates substrates on Ser/Thr residues.

PKB Inactivates Pro-Apoptotic Factor BAD

PKB inactivates the mitochondrial-linked pro-apoptotic factor BAD.

  • Phosphorylation of BAD leads to its inactivation and binding to 14-3-3 protein.
  • This allows Bcl-2 to remain active and promote cell survival.
  • Forkhead proteins are also regulated by PKB.

PTEN Removes PI3,4,5P3

PI(3,4,5)P3 is removed by the phosphatase and tumor suppressor PTEN, converting it back to PI(4,5)P₂.

PKB Cellular Targets

PKB has many cellular targets, including:

  • PDK1
  • eNOS
  • GSK-3β
  • mTOR
  • Iκκ NF-κB
  • BAD
  • 14-3-3
  • FKHR
  • Tuberin
  • Cyclin D (degraded via proteosome)

Regulation of Forkhead Localisation

Forkhead transcription factors are regulated by phosphorylation:

  • FKHRL1 is a forkhead family transcription factor.
  • 14-3-3 proteins bind to phosphoserine in a specific amino acid context.
  • Akt/PKB phosphorylates Forkhead, leading to its localization in the cytosol.

PKB Inhibits Caspases and Activates Translation

In anti-apoptotic pathways, PKB inhibits caspases and activates translation via S6 kinase.

PI 3-Kinases and Proliferation Signals

PI 3-kinases also generate proliferation signals:

  • Growth factor, cytokine, or hormone receptors, G protein-coupled receptors, and integrins activate PI 3-K.
  • PI(4)P, PI(4,5)P₂, and PI(3,4)P₂ are interconverted.
  • PTEN opposes PI 3-K.
  • PKB kinase domain is activated, leading to proliferation through p70S6K, GSK3, FHR-TF, p27Kip, and CyclinD1/Cdks.

TOR (Target of Rapamycin) for Signalling Growth

TOR is a key regulator of cell growth:

  • Growth/survival factors, oxygen, ATP, amino acids, and phosphatidic acid influence TOR.
  • TOR affects protein stability, ASK1, S6K1, 4E-BP1, transcription-factor translocation, translation elF4E, and ribosomal proteins.
  • This impacts apoptosis, ribosome biogenesis, and cap-dependent translation.

PI3K -TOR Regulation of Translation

PI3K and TOR regulate translation:

  • PDK1, IRS, PI3K, and PTEN are involved.
  • AKT phosphorylates downstream targets.
  • TSC1 and TSC2 regulate RHEB.
  • TOR, Raptor, and S6K1 regulate ribosomal protein translation and ribosome biogenesis.
  • 4E-BP1 regulates elF4E, affecting cap-dependent translation.

Additional Reading

  • BI1BEC1 Building blocks of life lecture - Responding to the environment: the basics of cell signalling.
  • Molecular Biology of the Cell (Alberts) 5th Ed Chpt 15 Mechanisms of Cell Communication pages 879-941.
  • Molecular Biology of the Cell (5th Ed) Pages 1060-1067 & 1101-1112.