Phosphorylation and Cell Signalling

A PTM (post-translational modification) is a covalent modification of a protein after translation.

  • They alter biochemical properties:

    • Protein conformation.

    • Enzyme activity (activation/deactivation).

    • Protein–protein interactions.

    • Subcellular localisation.

  • PTMs can be:

    • Co-translational (during synthesis)

    • Post-translational (after synthesis)

  • Reversible → allows dynamic cellular regulation.

Types of PTMs:

  • Phosphorylation – addition of phosphate (PO₄³⁻) group.

  • Methylation – addition of a methyl (–CH₃) group.

  • Acetylation – addition of an acetyl (–COCH₃) group.

  • Ubiquitylation – covalent attachment of ubiquitin protein.

Impact on Proteomic Complexity:

  • ~20,000 genes → ~100,000 transcripts (via alternative splicing).

  • PTMs expand proteomic diversity → >1,000,000 protein species.

  • PTMs amplify functional versatility:

    • Enable proteins to adopt multiple roles.

    • Facilitates rapid adaptation to physiological changes.

Phosphorylation: Biological & Pharmacological Significance:

  • Phosphorylation is the most studied PTM due to pharmacological relevance.

    • Kinases (enzymes adding phosphate groups):

      • Frequently mutated/overexpressed in disease (e.g. cancer, neurodegeneration, CVD, diabetes).

  • Kinase structure elucidation enabled:

    • Structure-based drug design (e.g. kinase inhibitors).

Phosphorylation as a Reversible Molecular Switch:

Mechanism:

  • Phosphorylation = addition of phosphate → often activates protein.

  • Dephosphorylation = removal of phosphate → often deactivates protein.

  • Acts as a molecular on/off switch.

Analogy: GTP-binding Proteins:

  • Similar to G-proteins cycling between:

    • GTP-bound (on) and GDP-bound (off) states.

  • Regulated by:

    • GAPs (GTPase-activating proteins).

    • GEFs (guanine nucleotide exchange factors).

Enzymes Involved:

  • Protein Kinases:

    • Transfer phosphate from ATP → target residues (Ser, Thr, Tyr).

    • Usually promote activation.

  • Protein Phosphatases:

    • Remove phosphate group (hydrolysis).

    • Usually promote deactivation.

  • Balance of kinase/phosphatase activity controls cellular signalling outcomes.

Functional Consequences of Phosphorylation:

Example: cAMP–PKA–CREB Pathway:

  • ↑ cAMP (e.g. via GPCR activation) binds to the regulatory subunits of Protein Kinase A (PKA).

  • Binding causes release of catalytic subunits, which translocate to the nucleus.

  • Catalytic subunits phosphorylate the transcription factor CREB at Serine-133.

  • Phospho-CREB binds to cAMP Response Elements (CREs) in target gene promoters.

  • Recruitment of coactivators (e.g., CBP/p300) → initiates transcription of cAMP-responsive genes.

❗Phosphorylation: CREB cannot bind effectively to DNA → no transcriptional activation.

General Outcomes:

  1. Conformational changes / complex formation
    → New or exposed binding sites enable protein–protein interactions.

  2. Indirect activation
    → Phosphorylation of one protein enables activation of another.

  3. Altered localisation
    → Phosphorylation directs proteins to new cellular compartments
    (e.g. RTK phosphorylation recruits signalling proteins to the membrane).

Protein Kinases:

Overview:

  • ~500 kinases in human genome = human kinome.

  • Types:

    • Ser/Thr kinases (majority).

    • ~90 Tyrosine kinases, incl. 56 RTKs (key in growth/differentiation).

Evolution & Structure:

  • Common ancestral kinase gene → conserved domains:

    • ATP-binding site – binds ATP for phosphate transfer.

    • Catalytic loop – mediates phosphate group transfer to substrate.

    • Activation loop – undergoes regulatory phosphorylation to control kinase activity.

  • Example: mTOR (Mammalian Target of Rapamycin)

    • Central regulator of cell growth, proliferation, and survival.

    • Often hyperactivated in cancer, driving uncontrolled cell division.

    • Inhibited by rapamycin, a small molecule that suppresses mTOR activity → therapeutic relevance in cancer and immunosuppression.

Activation by Second Messengers:

  • PKA → activated by cAMP.

  • PKC → activated by DAG (from PIP₂ cleavage by PLC-β).

  • CaM Kinase II → activated by Ca²⁺/calmodulin complex.

Protein Phosphatases:

  • Approximately 147 phosphatases identified — fewer than the number of protein kinases.

  • Structurally diverse → no single ancestral gene, unlike kinases.

  • Due to high diversity, phylogenetic relationships are difficult to resolve → often represented by “wheel-like” diagrams rather than linear trees.

  • Catalytic mechanism: all hydrolytically remove phosphate groups from substrates using water.

  • Generally more substrate-promiscuous than kinases → capable of dephosphorylating multiple targets across pathways.

Targeting Strategies:

  1. Module-Based:

    • Catalytic + targeting domains within same protein.

      • Targeting domain binds substrate motifs directly.

  2. SLiM-Mediated (Small Linear Motif):

    • Catalytic domain linked to short SLiM motif.

      • SLiM binds interaction domains (e.g. SH3).

  3. Regulatory Subunit-Assisted:

    • Catalytic subunit forms complex with regulatory subunit.

      • Regulatory subunit provides substrate specificity.

→ These ensure precise, context-specific dephosphorylation in cells.

Physiological Context of Insulin Signalling:

Animals face fluctuating feeding–fasting cycles → require efficient nutrient storage.

  • Insulin = peptide hormone from pancreatic β-cells (islets of Langerhans).

    • Secreted in response to ↑ blood glucose and other nutrients post-meal.

      • Main functions:

        • Promote uptake, utilisation, and storage of:

          • Glucose

          • Amino acids

          • Fatty acids

        • Target tissues: skeletal muscle, adipose tissue, liver.

    • Ensures excess nutrients are stored as:

      • Glycogen, proteins, lipids → energy reserves for fasting periods.

Activation of Insulin Signalling – Role of Tyrosine Phosphorylation:

Increased blood glucose levels lead to the release of insulin.

  • Insulin binds to Insulin Receptor (IR):

    • Homodimeric Receptor Tyrosine Kinase (RTK) on target cell membranes.

      • Binding of insulin causes a conformational change which activates the IR tyrosine kinase domain.

        • This leads to the autophosphorylation of specific tyrosine residues on the IR cytosolic domain.

          • This forms phosphotyrosine (pTyr) residues which are docking sites for proteins with SH2 domains.

IRS Proteins

  • Major SH2-binding adaptor proteins: IRS-1, IRS-2, IRS-3, and IRS-4.

    • The insulin receptor (IR) autophosphorylates and subsequently phosphorylates IRS proteins on tyrosine residues.

      • Phosphorylated IRS acts as a scaffold, recruiting and assembling multi-protein signalling complexes that propagate downstream insulin signalling.

Downstream Signalling Cascades & Metabolic Consequences:

PI3K/AKT Pathway (Primary Metabolic Route):

  1. IRS–PI3K binding:

    • IRS pTyr motifs recruit PI3K (phosphatidylinositol-3-kinase).

  2. PI3K activity:

    • Converts PIP2 → PIP3 (adds phosphate to 3′ position).

  3. PIP3 (second messenger):

    • Recruits PH-domain proteins to plasma membrane (e.g. AKT, PDK1).

  4. AKT (Protein Kinase B):

    • Activated by phosphorylation (via PDK1, mTORC2).

      • Central serine/threonine kinase drives the metabolic actions of insulin. AKT plays a crucial role in promoting cell survival and growth by inhibiting apoptotic processes and stimulating protein synthesis.

Major AKT-Mediated Effects:

  • ↑ Glucose uptake:

    • Promotes GLUT4 translocation to membrane (muscle/adipose).

  • ↑ Glycogen synthesis:

    • Activates glycogen synthase.

  • ↓ Gluconeogenesis:

    • Inhibits hepatic glucose output.

  • ↑ Lipogenesis:

    • Converts excess glucose → fatty acids/triglycerides.

  • ↑ Protein synthesis:

    • Stimulates anabolic pathways.

Other Pathway: MAPK Route:

  • IRS can recruit Grb2 → activates Ras–Raf–MAPK cascade.

    • Promotes growth, proliferation, survival.

      • However, PI3K/AKT is the main metabolic pathway.

Phosphorylation & Negative Feedback Regulation:


Phosphorylation also mediates self-limiting feedback loops to prevent overactivation.

Key Negative Feedback Mechanisms:

  • AKT Activation and mTORC1 Stimulation

    • AKT (Protein Kinase B) is activated downstream of PI3K in response to insulin signalling.

      • Activated AKT phosphorylates and activates mTOR Complex 1 (mTORC1) via interaction with the regulatory-associated protein RAPTOR.

    • mTORC1 phosphorylates and activates p70 S6 Kinase (S6K).

      • S6K then phosphorylates IRS proteins (particularly IRS-1) on serine residues, which:

        • Inhibits IRS activity, reducing its ability to transmit insulin signals.

        • Can also promote IRS degradation, further dampening the pathway.

    • mTORC1 also activates GRB10, an adaptor protein that:

      • Binds to the insulin receptor (IR) and inhibits IR–IRS interactions,

      • Providing additional negative feedback to limit insulin signalling.

  • AKT indirectly promotes the activity of PTEN (Phosphatase and Tensin Homolog) — a lipid phosphatase that:

    • Dephosphorylates PIP₃ → PIP₂,

      • Counteracting PI3K and thereby reducing AKT activation.

Insulin Resistance & Serine Phosphorylation:

Insulin resistance is a decreased tissue response to insulin, leading to impaired glucose uptake/utilisation.

  • Common in obesity and Type 2 Diabetes Mellitus (T2DM).

    • Type 1 DM: β-cell destruction → absolute insulin deficiency.

    • Type 2 DM: insulin resistance ± β-cell dysfunction → hyperglycaemia.

Inducers of Insulin Resistance:

  • Especially from visceral adipose tissue:

    • ↑ Free Fatty Acids (FFAs), glycerol.

    • Adipokines: leptin, adiponectin, resistin.

    • Pro-inflammatory cytokines: TNF-α, IL-6
      → Create chronic low-grade inflammation → activate IRS serine/threonine kinases.

IRS Kinase Categories:

  • Mediators of insulin signalling (feedback regulators): e.g. S6K.

  • Non-mediators (external stress/inflammatory kinases):

    • e.g. JNK, IKKβ, PKC, GSK3.

      • Strongly implicated in obesity-linked insulin resistance.

Mechanisms of IRS Inhibition:

  • Serine phosphorylation (vs activating tyrosine phosphorylation):

    • At PH or PTB domain sites: This phosphorylation can directly block IRS binding to the plasma membrane or the insulin receptor (IR), preventing its proper activation.

      • Other serine sites: Phosphorylation impairs IRS's critical scaffolding function, which can lead to:

        • Reduced recruitment of downstream effectors like PI3K.

        • IRS dissociation from the IR.

        • Enhanced IRS ubiquitylation and subsequent proteasomal degradation.

    • Cumulative effect → pathway inhibition, ↓ glucose uptake lead to hyperglycaemia and diabetic symptoms (fatigue, thirst, blurred vision).

Retinoblastoma (Rb) Overview:

Tumour suppressor protein, crucial for cell cycle control.

  • Regulates G1 → S phase transition.

    • Activity determined by phosphorylation status:

      • Hypophosphorylated Rb → active

      • Hyperphosphorylated Rb → inactive

  • Mutations or deregulation can lead to uncontrolled cell proliferation and tumour development.

Rb and E2F Interaction:

E2F transcription factor:

  • Drives transcription of genes required for DNA replication and S-phase entry.

    • Active E2F → promotes cell cycle progression and cell division.

  • Active Rb (hypophosphorylated):

    • Binds E2F → inhibits transcription of S-phase genes → prevents cell division.

  • Inactive Rb (hyperphosphorylated):

    • Releases E2F → allows transcription of S-phase genes → promotes cell division.

Phosphorylation and Cell Cycle Control:

  • Cyclin-CDK complexes mediate Rb phosphorylation:

    • Early G1 → Rb is mostly hypophosphorylated → active.

    • Mid to late G1 → sequential phosphorylation by different cyclin-CDK complexes.

      • Sufficient growth factor signals lead to hyperphosphorylation causing Rb to become inactive.

  • Post-translational modifications:

    • Phosphorylation at multiple sites induces specific Rb conformations.

      • Each conformation allows interaction with different proteins, regulating multiple pathways.

Anti-Growth Signals (TGF-β Pathway):

  • TGF-β = anti-proliferative factor.

    • Mechanism:

      • TGF-β binds receptor, leading to the activation of the signalling cascade which then induces p15 and p21.

        • p15/p21 inhibit cyclin-CDK complexes which prevents Rb phosphorylation.

          • Rb remains hypophosphorylated meaning it is active, and so the cell cycle arrests.

Rb Structure and Protein Interactions:

  • Key domains:

    • Pocket domain → binds E2F transactivation domain.

    • RBC domain → binds both E2F and DP subunits.

    • LxCxE binding cleft → binds other regulatory proteins; targeted by viral oncoproteins.

  • E2F structure: heterodimer of E2F + DP subunits.

  • Active Rb:

    • Open conformation.

    • Pocket and RBC domains bind E2F which inhibits S-phase gene expression.

  • Inactive Rb:

    • Hyperphosphorylation leads to a closed conformation.

    • Releases E2F, leading to the transcription of S-phase genes.

Viral Deregulation:

  • Human papillomavirus (HPV):

    • E7 protein binds LXCXE cleft.

    • Causes Rb degradation → uncontrolled E2F activity → promotes cervical cancer.