Signal Transduction: Tyrosine Kinase Signaling, JAK-STAT, RTKs, and Signal Termination

Introduction to Cytokine Receptors (CRs)

  • Definition and Function: Cytokines are critical signaling proteins for the growth, differentiation, and activation of cells, playing a central role in the immune system. Examples include: Growth hormone, Prolactin, Interleukins, Erythropoietin (EPO), and Interferon

  • Primary Signaling Module (JAK-STAT): Cytokine receptors primarily signal through the JAK-STAT module.

    • JAK : A tyrosine kinase that initiates the signal. JAK stands for Janus Kinase.

    • STAT: A transcription factor that regulates gene expression.

  • Initiation via Ligation: Activation of cytokine receptors (and Receptor Tyrosine Kinases) typically requires the formation of a dimer consisting of two chains.

  • Mechanisms of Dimerization:

    • Ligand-Induced Dimerization: The binding of a ligand causes monomeric receptor chains to homodimerize.

    • Pre-formed Dimers: In some cases, the receptor exists as a pre-formed dimer; ligand binding triggers a structural/conformational change that brings the cytosolic tails together.

Signal Initiation and JAK-STAT Pathway Mechanism

  • JAK-Receptor Interaction: Each receptor chain is bound to a specific JAK kinase (e.g., JAK1, JAK2, JAK3, or Tyk2). Before ligation, these kinases are situated too far apart to interact.

  • Activation Sequence:

    1. Receptor ligation brings the JAKs into close proximity.

    2. Trans-phosphorylation: Each JAK phosphorylates the other (auto-phosphorylation on the activation lip), activating its kinase function.

    3. Receptor Phosphorylation: The activated JAKs then phosphorylate specific tyrosine residues on the receptor chains.

  • The SH2 Domain: Phosphorylated receptor chains create docking sites for proteins with a phosphotyrosine-binding motif, specifically the SH2 domain.

  • STAT Recruitment and Activation:

    1. STAT monomers are recruited from the cytosol to the phosphorylated receptor via their SH2 domains.

    2. JAKs phosphorylate the STAT monomers on tyrosine residues.

    3. Phosphorylation causes STAT to dissociate from the receptor.

  • STAT Dimerization and Nuclear Entry:

    1. Once phosphorylated, STATs undergo self-interaction: the SH2 domain of one STAT monomer binds to the phosphotyrosine of another STAT monomer (forming a dimer).

    2. Dimerisation reveals a Nuclear Localization Sequence (NLS).

    3. The STAT dimer enters the nucleus, binds to specific DNA motifs, and regulates gene expression by recruiting master transcription factors and modifying chromatin.

Receptor Tyrosine Kinases (RTKs)

  • Overview: RTKs regulate cell survival, proliferation, and metabolism. Mutations in RTKs are frequently associated with cancer development.

  • Difference from Cytokine Receptors: While cytokine receptors rely on recruited JAK kinases, RTKs have the tyrosine kinase domain intrinsic to the receptor protein itself.

  • RTK Initiation: Ligand binding triggers a structural change/dimerisation. The internal kinase domains activate each other via trans-phosphorylation and then phosphorylate other tyrosine residues on the receptor tails.

RTK Signal Relay and Effector Pathways

  • Docking Site Specificity: The sequences adjacent to the phospho-tyrosine residue (pYpY) determine which SH2-containing protein will bind.

  • Major Downstream Pathways:

    1. STAT Pathway: Directly activates transcription factors.

    2. Phospholipase C (PLC) Pathway: Elevation of cytosolic Ca2+Ca^{2+}.

    3. PI-3 Kinase Pathway: Activates Protein Kinase B (PKB/AKT) for cell survival and metabolism.

    4. MAP Kinase (MAPK) Pathway: Drives cell division and growth.

The Phospholipase C (PLC) and PI-3 Kinase Pathways

  • PLC gamma vs PLC beta:

    • PLCβ\beta is used in GPCR signaling and is activated by G-proteins.

    • PLCγ\gamma is used in RTK/CR signaling. It contains an SH2 domain that allows it to bind directly to the phosphorylated receptor. Once bound, it is phosphorylated by the receptor/JAK and brought close to its substrate, PIP2PIP_2, which it cleaves into IP3 and DAG.

  • PI-3 Kinase Pathway:

    1. PI-3 Kinase is recruited via its SH2 domain to the phosphorylated receptor.

    2. It phosphorylates the 3rd position of the inositol ring of PIPPIP or PIP2PIP_2, generating PI-3 phosphates (e.g., PIP3PIP_3).

    3. Protein Kinase B (PKB/AKT) Activation: PKB binds to PI-3 phosphates via its PH domain, which unmasks its kinase activity.

    4. Kinases PDK1 and PDK2 then phosphorylate PKB for full activation.

    5. Function: Active PKB regulates cell survival (inhibiting cell death) and glucose uptake. Dysregulation is a hallmark of cancer.

The MAP Kinase (MAPK) Pathway

  • Overview: Almost all RTKs and CRs activate this pathway to drive cell division.

  • Ras G-Protein: Path initiation requires Ras, a monomeric G-protein.

    • Ras is activated by Sos, which functions as a GEF (Guanine Nucleotide Exchange Factor).

    • Grb2 acts as an adaptor protein (SH2 domain binds the receptor; SH3 domains bind Sos).

  • Kinase Cascade:

    1. Activated Ras recruits and activates Raf (a MAP Kinase Kinase Kinase).

    2. Raf phosphorylates MEK (a MAP Kinase Kinase).

    3. MEK phosphorylates MAPK at both Thr and Tyr residues to become activated.

    4. Activated MAPK can dimerise and phosphorylate cytosolic substrates or transcription factors in nucleus.

  • Substrate Diversity: Raf and MEK have very few substrates, but MAPK has over 170 known targets, including transcription factors that trigger cell division.

  • Clinical Relevance: Mutations that lock Ras in a "GTP-bound" state or mutations in Raf that activate its kinase activity are very common in cancers.

Signal Termination (Turning the Signal OFF)

  • A. Turning off the Kinase: Phosphatases remove the activating phosphates from the kinases.

    • SHP1: A phosphatase with an SH2 domain that binds to phosphorylated cytokine receptors and dephosphorylates the JAK kinase.

  • B. Turning off the Receptor:

    • Phosphatases: Dephosphorylate the receptor tails directly.

    • Receptor Internalization: Receptors are moved from the membrane to lysosomes for degradation or recycled.

    • SOCS (Suppressors of Cytokine Signaling): Proteins that target phosphorylated receptors for degradation and block binding sites for other signaling molecules. SOCS knockout mice also show severe inflammatory phenotypes.

  • C. Turning off the Second Messenger:

    • PTEN: A phosphatase that removes the phosphate from PI-3 phosphates (the opposite of PI-3 Kinase).

    • Cancer Link: The PTEN gene is frequently deleted in cancers, leading to uncontrolled PKB/AKT signalling and cell proliferation.

Questions & Discussion

  • Q: How does ligand-receptor interaction trigger signalling from RTKs and cytokine receptors?

    • A: By inducing a structural change (often dimerisation or reorientation of a pre-formed dimer) that brings the internal or associated cytoplasmic kinase domains close enough to cross-phosphorylate each other.

  • Q: How does signaling from RTKs and cytokine receptors differ? How is it similar?

    • A: Similarity: Both use tyrosine phosphorylation, SH2 domains for docking, and activate similar downstream paths (MAPK, PI3K, PLC). Difference: RTKs have intrinsic kinase activity; CRs utilize non-covalently associated JAK kinases.

  • Q: Name the four major pathways triggered by RTKs and CRs and describe their relay/amplification.

    • A: 1. JAK-STAT (Direct relay to transcription), 2. MAPK (Ras-Raf-MEK-MAPK cascade), 3. PI3K/AKT (Lipid signaling via PH domains), 4. PLCγ\gamma (Lipid cleavage to Ca2+Ca^{2+} and PKC).

  • Q: Explain three mechanisms of signal shutdown with examples.

    • A: 1. Kinase dephosphorylation (SHP1), 2. Receptor degradation (SOCS), 3. Second messenger removal (PTEN).

  • Q: Give two examples of how dysregulation of these systems leads to disease.

    • A: 1. Ras/Raf mutations in cancer (melanoma), 2. PTEN deletion leading to uncontrolled proliferation.