Signal Transduction Biochemistry

Signal transduction is the process by which extracellular signals (hormones, growth factors, cytokines, neurotransmitters) are detected at the cell surface and converted into intracellular biochemical responses. Understanding these pathways is fundamental to pharmacology, oncology, and immunology.

Principles of Signal Transduction

Signal transduction involves: (1) Ligand-receptor binding (high affinity, saturable, specific); (2) Signal amplification (one receptor activates many second messengers); (3) Integration (multiple signals converge and diverge); (4) Termination (phosphatases, GTPase activity, receptor desensitisation, endocytosis). Receptors are classified by their location and mechanism: plasma membrane receptors (ion channel-linked, G protein-coupled, enzyme-linked) and intracellular receptors (nuclear — steroid hormones, thyroid hormone, vitamin D).

Receptor Tyrosine Kinases (RTKs)

RTKs are single-pass transmembrane proteins with an extracellular ligand-binding domain and an intracellular tyrosine kinase domain. Examples: EGFR (ErbB1), HER2 (ErbB2), PDGFR, VEGFR, FGFR, insulin receptor. Mechanism: Ligand binding (EGF to EGFR) induces receptor dimerisation. Dimerisation enables transphosphorylation — each kinase phosphorylates specific tyrosine residues on its partner. Phosphotyrosine residues serve as docking sites for SH2 domain-containing adaptor proteins (Grb2, Shc, PI3K regulatory subunit p85) and phospholipases (PLCγ). Activated RTKs are internalised via clathrin-mediated endocytosis and either recycled or degraded in lysosomes — a mechanism of signal attenuation. RTK mutations are frequent in cancer: EGFR activating mutations (lung adenocarcinoma, responsive to erlotinib/gefitinib); HER2 amplification (breast cancer, trastuzumab/pertuzumab); KIT and PDGFRA mutations (GIST, imatinib).

Ras-MAPK Cascade

Ras is a small GTPase (monomeric G protein) — active when GTP-bound, inactive when GDP-bound. Intrinsic GTPase activity slowly hydrolyses GTP to GDP; activated by GTPase-activating proteins (GAPs); reactivated by guanine nucleotide exchange factors (GEFs, e.g., SOS). Following RTK activation: Grb2 (SH2 + two SH3 domains) bridges phosphorylated RTK to SOS (a GEF). SOS catalyses GDP → GTP exchange on Ras → Ras-GTP active.

Ras-GTP activates Raf (a serine/threonine kinase, MAP3K). Raf phosphorylates MEK1/2 (MAP2K). MEK phosphorylates ERK1/2 (MAPK) on both threonine and tyrosine. Active ERK enters the nucleus and phosphorylates transcription factors (Elk-1, c-Fos, c-Jun components of AP-1), driving proliferation, differentiation, and survival gene expression. ERK also phosphorylates cytoplasmic targets (RSK, MNK). Negative feedback: ERK phosphorylates SOS (inhibiting GEF activity), Raf (reducing its activity), and induces DUSP (dual-specificity phosphatases) that dephosphorylate ERK.

Ras mutations (KRAS G12D, G13D, G12V — most common; NRAS, HRAS) constitutively activate Ras by impairing GTPase activity → uncontrolled MAPK signalling. Found in >30% of all cancers (pancreatic ductal adenocarcinoma ~95%, colorectal ~45%, NSCLC ~30%). KRAS was long considered "undruggable" — AMG 510 (sotorasib, specifically targets KRAS G12C) is the first approved KRAS inhibitor.

PI3K-Akt-mTOR Pathway

Class I PI3K is a heterodimer (regulatory p85 + catalytic p110). Activated by RTKs (p85 SH2 binds phosphotyrosine), Ras-GTP, and GPCRs. PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) at the 3-OH position of the inositol ring → phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 recruits PDK1 and Akt to the plasma membrane via their PH domains. PDK1 phosphorylates Akt at Thr308; mTORC2 phosphorylates Akt at Ser473 (full activation). PTEN is the major negative regulator — a 3-phosphatase that reverses PIP3 → PIP2. PTEN is the second most frequently mutated tumour suppressor in human cancer.

Active Akt phosphorylates: GSK-3β (inactivation → glycogen synthesis); FOXO transcription factors (cytoplasmic sequestration → reduced apoptosis gene expression); MDM2 (enhanced p53 degradation → reduced apoptosis); BAD (inhibition → survival); TSC2 (inactivation) → mTORC1 activation.

mTORC1 (mechanistic target of rapamycin complex 1): phosphorylates S6K1 (activates ribosomal protein S6, promoting ribosome biogenesis and translation) and 4E-BP1 (phosphorylation releases eIF4E, promoting cap-dependent mRNA translation). Net effect: cell growth and proliferation. Rapamycin (sirolimus): allosteric mTORC1 inhibitor (forms complex with FKBP12 → binds mTOR). Everolimus: derivative, used in renal cell carcinoma, breast cancer, transplant immunosuppression.

PI3K-Akt-mTOR dysregulation: PIK3CA (p110α) activating mutations in breast, colorectal, endometrial cancers; PTEN loss in glioblastoma, prostate cancer; Akt amplification; TSC1/2 loss (tuberous sclerosis → mTORC1 hyperactivation → hamartomas). Alpelisib (PI3Kα inhibitor) + fulvestrant is approved for PIK3CA-mutant ER+ breast cancer.

JAK-STAT Pathway

Cytokines (interferons, interleukins, EPO, G-CSF) bind receptors associated with Janus kinases (JAK1/2/3, TYK2). Cytokine binding induces receptor homodimerisation (or heterodimerisation) → JAK transphosphorylation and activation → JAKs phosphorylate STAT (signal transducer and activator of transcription) proteins (STAT1-6) on a specific tyrosine → STAT dimerisation via reciprocal SH2-pY interactions → nuclear translocation → STAT-responsive gene transcription. Negative regulation: SOCS proteins (suppressor of cytokine signalling) are JAK-STAT target genes that feedback-inhibit the pathway (SOCS bind JAKs or phosphotyrosines, competing with STATs, and recruit ubiquitin E3 ligases). Also: SHP phosphatases dephosphorylate JAKs and STATs; STATs are dephosphorylated in the nucleus by nuclear phosphatases; PIAS proteins SUMOylate STATs.

Key biological roles: IFN-α/β (STAT1/2 → ISGs — antiviral immunity), IFN-γ (STAT1 → MHC-II induction, macrophage activation), IL-6 (STAT3 → acute phase response, IL-6 paradox in inflammation and cancer), EPO (STAT5 → erythropoiesis, JAK2 V617F — polycythaemia vera). JAK inhibitors (jakinibs): ruxolitinib (JAK1/2 — myelofibrosis, PV), tofacitinib (JAK1/3 — RA, IBD, psoriatic arthritis), baricitinib (JAK1/2 — RA, COVID-19 — reduces inflammation-driven cytokine storm).

Second Messengers: cAMP

cAMP is synthesised from ATP by adenylyl cyclase (AC), activated by Gs-coupled GPCRs (beta-adrenergic receptors, glucagon receptor, TSH receptor) and inhibited by Gi-coupled receptors (alpha2-adrenergic, M2 muscarinic). cAMP is degraded by phosphodiesterases (PDEs) — therapeutic targets: sildenafil (PDE5 inhibitor → smooth muscle relaxation → vasodilation — erectile dysfunction, pulmonary hypertension), theophylline (PDE3/4 inhibitor → bronchodilation).

cAMP activates PKA: 2 cAMP bind each R subunit (4 total) → R subunits dissociate → C subunits active. PKA phosphorylates: (a) Glycogen phosphorylase kinase → glycogenolysis; (b) PFK-2/FBPase-2 → reduced F2,6-BP → reduced glycolysis; (c) Pyruvate kinase → reduced glycolysis; (d) Hormone-sensitive lipase → lipolysis; (e) CREB (cAMP response element binding protein) → CRE-driven gene transcription (e.g., tyrosine hydroxylase, gluconeogenic enzymes). Cholera toxin: ADP-ribosylates Gsα, preventing GTPase activity → Gsα constitutively active → AC maximally active → massive cAMP → massive PKA → phosphorylation of CFTR → Cl- and water secretion → profuse watery diarrhoea. Pertussis toxin: ADP-ribosylates Giα, locking it in inactive GDP-bound form → prevents Gi inhibition of AC.

Second Messengers: IP3 and DAG

PLCβ (activated by Gq-coupled receptors — M1 muscarinic, alpha1-adrenergic, angiotensin AT1) or PLCγ (activated by RTKs) cleaves PIP2 → inositol-1,4,5-trisphosphate (IP3) + diacylglycerol (DAG). IP3: water-soluble, diffuses to ER, binds IP3 receptors (IP3R, Ca2+-release channels) → Ca2+ release from ER into cytoplasm. Ca2+ acts as a second messenger: activates calmodulin (CaM) → CaM-kinases (CaMKII — CREB phosphorylation, glycogen metabolism), calcineurin (phosphatase → NFAT dephosphorylation → T-cell cytokine gene transcription — target of cyclosporin and tacrolimus), troponin C (muscle contraction), calmodulin-dependent MLCK (smooth muscle contraction). DAG: lipid-soluble, stays in membrane; activates protein kinase C (PKC) — phosphorylates diverse substrates promoting proliferation, cytokine production, and neurotransmitter release. PKC also activated by tumour-promoting phorbol esters (e.g., TPA, which mimic DAG).