BI2BC45 Cells and Immunity - Pathways regulating survival, growth and proliferation - Part 1

Pathways Regulating Survival, Growth, and Proliferation

Topics Covered

This lecture and the next will cover pathways that control:

Cell proliferation
Cell growth
Cell division
Cell survival

Later lectures will explore how cell survival balances apoptotic signals and how cell proliferation influences cell cycle control.

Key Concepts in Cell Signalling

Specific molecular recognition: Cells must be able to specifically recognize signals from their environment.
Molecular switches: These control signaling pathways. Examples include G proteins and phosphorylation.
Signal amplification: The initial signal is amplified to produce a larger downstream effect.
Second messengers: These are small molecules that relay signals within the cell.

Cell Signalling

Cells are constantly bombarded with signals and must interpret them to determine appropriate responses such as:

Survival
Division
Differentiation
Apoptosis (programmed cell death)
Senescence (cellular aging)

Coincidence Detection

Cells often require multiple signals to trigger a response. A response logic gate analogy is used; if either signal 1 or signal 2 is blocked, there is no output.

Integration of Multiple Signals

Different responses require integration of various signals:

Apoptosis vs. Cell Division (Ras/MAP kinase pathway): Strong Ras/MAP kinase signaling can lead to cell division, while weak signaling may result in apoptosis.
Cell Division vs. Cell Survival (PI 3-kinase/PKB/Akt pathway): Weak PI 3-kinase signaling may trigger cell division, but strong signaling promotes cell survival.
Integrated Response (Ras/MAP kinase & PI 3-kinase/PKB/Akt): Strong signaling from both pathways is required for cell division and cell survival.

Signalling proliferation: RAS/MAP kinase (ERK) & cell cycle

Cell Cycle Overview

The cell cycle consists of four phases:

G1 (Gap 1): Cell growth and preparation for DNA replication.
S (Synthesis): DNA replication.
G2 (Gap 2): Further growth and preparation for mitosis.
M (Mitosis): Cell division.

Cells can also enter a quiescent state (G0) when serum-starved.

The cell cycle has checkpoints:

G1 checkpoint
S checkpoint
G2 checkpoint
Metaphase checkpoint

Checkpoints

G1 checkpoint: Is the environment favorable?

Cyclin/CDK Control Points

Cyclin/CDK complexes regulate the cell cycle:

Cyclin D/CDK4/CDK6
Cyclin E/CDK2
Cyclin A/CDK2
Cyclin A/CDK1
Cyclin B/CDK1

Protein-Tyrosine Kinase Receptor – Ras – MAP Kinase Cascade

This is a growth factor-activated signaling pathway used by many PTK receptors, leading to changes in gene expression and cell cycle progression. The pathway is often activated in various cancers.

Receptor Phosphorylation

Receptors become phosphorylated upon ligand binding. This initiates downstream signaling events.

Phosphorylation

Phosphorylation is the enzyme-mediated transfer of a phosphate group ( $PO_4^{2-}$ ) from ATP to another molecule (e.g., proteins, lipids, sugars, DNA). Kinases mediate the addition of phosphate, while phosphatases catalyze its removal.

Phosphorylation Cycle

The process is rapid and reversible:

$Protein + ATP \xrightarrow{Kinase} Protein-P + ADP$

$Protein-P \xrightarrow{Phosphatase} Protein$

Protein Kinases

Two main classes exist:

Protein Serine/Threonine kinases
Protein Tyrosine kinases

Tyrosine Autophosphorylation

Tyrosine autophosphorylation of receptors leads to the recruitment of signaling proteins and subsequent cellular responses.

Effector Systems

Proteins that bind to and are activated by the ligand-stimulated receptor are effector systems. These enzymes determine intracellular signals and the response to a stimulus.

Adaptor Domains

Adaptor domains in signaling proteins (e.g., PH and SH2 domains) bind to specific target sequences or molecules.

Adaptor Proteins

Adaptor proteins organize signal transduction complexes through interactions like:

SH2 or PTB domains with phosphorylated proteins
SH3 domains with proline-rich sequences
PH domains with membrane phospholipids

Signalling Domains

Signaling domains (~100 amino acids) are found in many proteins.

Examples include:

Src family PTKs
Grb2/Sem-5/Drk
Ras-GAP
SOS/Cdc24
Shc
Abl/BCR
PLCy
p85a
p91/STAT

SH2 Domains

SH2 domains are independently folding protein modules (~100 amino acids long) found in many signaling molecules. They regulate the activation of signaling proteins via protein-tyrosine kinases.

SH2 domains bind to phosphorylated tyrosine residues within a specific sequence context (e.g., Y(P)xxM for PI 3-kinase, Y(P)xNx for Grb2).

These domains mediate the formation of signaling complexes between activated protein-tyrosine kinase receptors and downstream generators of second messenger molecules.

Consequences of Domain-Mediated Translocation to Receptor

Translocation
Phosphorylation
Conformational change

Molecular Switches

Protein phosphorylation: Regulated by kinases and phosphatases.
G proteins: Guanosine triphosphate (GTP)-binding proteins.

Signaling

A) SIGNALING BY PHOSPHORYLATION

SIGNAL IN -> APP -> ON

SIGNAL IN -> OFF

B) SIGNALING BY GTP-BINDING PROTEIN

SIGNAL IN -> GPP -> ON

SIGNAL IN -> OFF

Activation of Receptor Tyrosine Kinase and Downstream Signaling

This involves:

Ligand binding
Activation of protein-tyrosine kinase activity
Ras G protein molecular switch
Coupling of signal via SH2/SH3 domains
Activation of SOS, a G protein
Protein kinase cascade (Raf-1, MEK, MAPK)
Altered activity of transcription factors (c-Fos, c-Jun, AP1)

Ras G Protein Molecular Switch

Ras alternates between an inactive GDP-bound state and an active GTP-bound state. Guanine nucleotide exchange factors (GEFs) activate Ras by promoting the exchange of GDP for GTP, while GTPase-activating proteins (GAPs) inactivate Ras by stimulating GTP hydrolysis.

Protein-Tyrosine Kinase Receptor – Ras – MAP Kinase Cascade

A series of kinases amplifies the signal as it progresses into the cell.

Signal Amplification Cascade

This cascade converts a single ligand-receptor interaction into a large amount of activity inside the cell. It depends on the catalytic ability of each enzyme in the cascade to produce or activate more than one product molecule (e.g., the Ras–MAP kinase cascade).

Amplification Example

One activated Ras molecule activates one Raf-1 molecule (no amplification).
Each Raf-1 molecule phosphorylates 10 MEK molecules.
If each MEK and downstream kinase phosphorylates 10 substrate molecules, the total amplification is 10,000-fold.

$1 \rightarrow 1 \rightarrow 10 \rightarrow 100 \rightarrow 1000 \rightarrow 10000$

Ras-MAP Kinase Pathway

One endpoint is the activation of transcription factors, resulting in new gene and protein expression.

Cyclin/CDK Control Points in Cell Cycle

Cyclin D/CDK4/CDK6 (G1)
Cyclin E/CDK2 (G1/S)
Cyclin A/CDK2 (S)
Cyclin A/CDK1 (G2/M)
Cyclin B/CDK1 (M)

The Ras-MAPK Pathway Regulates Myc Expression

Mitogens activate the Ras-MAPK pathway, leading to increased Myc expression and subsequent effects on:

Cyclin D gene
SCF subunit gene
E2F gene

This promotes G1-Cdk activation (cyclin D-Cdk4), G1/S-Cdk activation (cyclin E-Cdk2), Rb phosphorylation, and increased E2F activity, ultimately leading to entry into S phase.

Growth Factors Regulate Cell Cycle Through Cyclin D Kinases

Mitogens stimulate Cyclin D/CDK4/CDK6, which phosphorylates Rb, releasing E2F and promoting transcription of S-phase genes.

INK4A inhibits CDK4/CDK6, while differentiation and apoptosis inhibit G1 and promote S-phase.

Too Much Myc Protein

Excessive Myc production can cause cell cycle arrest or apoptosis via p53 activation.

p53, MYC and E2F Effector Pathways

p53 promotes apoptosis, cell-cycle arrest, and DNA/nucleotide biogenesis.

MYC promotes cell-cycle progression, metabolism, and ribosome biogenesis.

E2F promotes cell-cycle progression, DNA/nucleotide biogenesis.

Cell Cycle Targets of Ras/Raf Signal Transduction Pathways

Ras and Raf pathways target Jun, CycD, cdc25, CycD, cdk4, CycE, cdk2, Myc, CycE, E2F, and Rb, leading to S phase.

Pathway Illustration

The pathway illustrates key signaling concepts:

Molecular recognition
Phosphorylation
Adaptor proteins
Protein-protein interactions
Molecular (G-protein) switch
Amplification cascade

Further Studies

Next lectures will cover growth and survival pathways regulated by TOR and PI 3-kinase, integrating with Ras-MAPK signals.