05 - Signaling mechanisms regulated by receptor tyrosine kinases P1

Transclude of Auto--and-trans-phosphorylation.ppsx

TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Cell Signaling::05 - Signaling mechanisms reg by RTK P1

1. Insulin – Quaternary Structure

Insulin Assembly State

$\beta$-cells release hexameric ${\text{Zn}}^{2+}$-insulin into the extracellular space, but monomeric ${\text{Zn}}^{2+}$-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be ${\text{Zn}}^{2+}$ concentration-dependent.

$\text{monomer}\to \text{dimer}\to \text{hexamer (secretory vesicles)}$

• The ${\text{Zn}}^{2+}$-binding properties of albumin improve the dissociation of ${\text{Zn}}^{2+}$-insulin into subunits after exocytosis.
• Dimer dissociation (loss of the interfacial $\beta$-sheet and solvation of the hydrophobic core) is a prerequisite for insulin to bind to its cellular receptor.
• The insulin receptor is a large protein that binds to insulin and carries its message inside the cell.

What is the biologically active form of insulin?

Monomeric ${\text{Zn}}^{2+}$-free insulin. $\beta$-cells secrete hexameric ${\text{Zn}}^{2+}$-insulin, but dimer dissociation (facilitated by albumin’s ${\text{Zn}}^{2+}$-binding properties) is required for receptor binding.

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Dimer dissociation, involving loss of the interfacial {1:$\beta$-sheet} and solvation of the {2:hydrophobic core}, is a prerequisite for insulin to bind to its cellular receptor.

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2. Overview of Receptor Types

Receptor Type	Mechanism	Second Messenger / Effector
Gated ion channel	Opens/closes in response to ligand concentration or membrane potential	Ion flux
Serpentine receptor (GPCR)	Ligand → R → G protein → Enzyme (Enz)	Intracellular second messenger X
Receptor with no intrinsic enzyme activity	Interacts with cytosolic protein kinase → gene-regulating protein (directly or via kinase cascade)	Gene expression change
Receptor enzyme (RTK, receptor guanylyl cyclase)	Ligand binding stimulates intracellular enzyme domain	Phosphorylation cascade
Steroid receptor	Steroid binds nuclear receptor → regulates gene expression	mRNA / Protein

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3. Receptor Tyrosine Kinases (RTK)

3.1 General Structure

RTK General Architecture

RTKs are generally single transmembrane polypeptide chains that cross the cell membrane once ($\alpha$-helix). The intracellular part contains a tyrosine kinase domain, inactive in the absence of ligand. The C-terminal segment is rich in Tyr (multiple copies).

Examples of RTK ligands: EGF (epidermal growth factor), Insulin, PDGF (platelet-derived growth factor).

3.2 Kinase and Phosphatase Definitions

Key Enzyme Definitions

• KINASE: enzyme that transfers a phosphoryl group from a nucleotide tri-phosphate to an acceptor molecule (phosphorylation)
  • Tyr kinase activity: enzymatic phosphorylation of Tyrosine residues ($-\text{OH}$) by ATP
• PHOSPHATASE: enzyme that removes a phosphoryl group from a phosphate ester, with water as the attacking species (dephosphorylation)
• The phosphate group adds negative charges to the individual amino acid residue and to the whole polypeptide.

What is the difference between a kinase and a phosphatase?

A kinase transfers a phosphoryl group from a nucleotide triphosphate to an acceptor molecule (phosphorylation). A phosphatase removes a phosphoryl group from a phosphate ester using water (dephosphorylation).

3.3 Activation Mechanism

RTK Activation by Ligand-Induced Dimerization

In the presence of ligand(s) bound to the extracellular domain, the RTK becomes an activated dimer. The kinase domains come into contact and are activated by trans/auto-phosphorylation, resulting in phosphorylation of specific Tyr residues in the intracellular part of the receptor next to the kinase domain.

• Signal transduction is mediated by receptor activation due to hormone-induced dimerization.
• Covalent trans/auto-phosphorylation of specific Tyr residues allows signal activation even after hormone dissociation.

Signaling Deactivation

Signaling is deactivated by:

1. Dephosphorylation of the Tyr residues (PTP, phosphatase)
2. Additional phosphorylation of intracellular residues of Ser and Thr (by cAMP-processes)

Anki cloze

RTK signaling is deactivated by {1:dephosphorylation of Tyr residues by PTP} and by {2:additional phosphorylation of intracellular Ser and Thr residues by cAMP-processes}.

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4. Intracellular Signal Transduction – Docking Proteins

SH2 Domain Transducers

The phosphorylated Tyr residues become binding sites for signaling partner proteins (transducers) that contain SH2 (Src homology 2) domains. The transducers also become phosphorylated by the kinase or are activated by conformational changes, and start the intracellular signal transduction.

4.1 Types of Docking Proteins (Transducers)

Protein	Category	Function
GRB2 (Growth-factor-receptor-binding protein 2)	Without catalytic activity	Recruitment of other signal proteins
Shc (Src Homologous and Collagen Protein)	Without catalytic activity	Recruitment of other signal proteins
PLC-γ (Phospholipase C)	Enzyme	Phospholipase activity
c-Src (Cellular SARC)	Enzyme	Tyr-protein kinase
GAP (GTPase Activating Protein)	Enzyme	Ras-GTPase activator
SH-PTP1 and PTP2	Enzyme	P-protein phosphatase
PI3K	Enzyme	Phosphoinositide kinase

Key Adapter Complex

Grb2 is an adapter protein that exists in a complex with SOS (Son of Sevenless), a guanyl nucleotide exchange factor that promotes GDP/GTP exchange on the small G protein Ras. $\text{GTP-Ras}=\text{active form}$

What is the role of SOS in RTK signaling?

SOS (Son of Sevenless) is a guanyl nucleotide exchange factor (GEF) that promotes GDP→GTP exchange on Ras, converting it to its active GTP-bound form. SOS is recruited to the membrane via its association with the adapter protein Grb2.

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5. Ras – Activation Cycle and Oncogenic Mutations

Ras GTPase Cycle

• Inactive Ras: GDP-bound
• Input signal stimulates GDP→GTP exchange (intrinsic GTPase activity)
• Active Ras: GTP-bound → activates effector → output signal
• Signal terminates via intrinsic GTPase activity of Ras (further enhanced by GAP)
• Oncogenic Ras proteins are blocked at the active (GTP-bound) state → signal remains on

Oncogenic Ras

Oncogenic Ras proteins are blocked at the GTP-bound active state, meaning the signal remains permanently on. This is a common mechanism of uncontrolled cell proliferation in cancer.

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Oncogenic Ras proteins are blocked at the {1:GTP-bound} state, meaning the intrinsic {2:GTPase} activity is impaired and the signal remains permanently active.

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6. The MAPK Cascade

Three-Tiered Kinase Cascade

A cascade in which each kinase activates the next by phosphorylation:

Level	Kinase	Activation
MAPKKK	Raf	Activated by membrane-bound GTP-Ras; activation is a multistep process
MAPKK	MEK	Phosphorylated by Raf on two Ser residues; is a Ser/Thr + Tyr protein kinase
MAPK	ERK (extracellular-signal-regulated kinase)	Phosphorylated by MEK on a Thr and a Tyr residue

Raf-1 Activation Detail

Raf-1 activation is a multistep process involving:

• Dephosphorylation of inhibitory sites by protein phosphatase 2A (PP2A)
• Phosphorylation of activating sites by PAK ($p21^{rac/cdc42}$-activated kinase), Src-family kinases, and yet unknown kinases

Scaffold Organization

The whole three-tiered kinase cascade is scaffolded — substrates exist in both the cytosol/cytoskeleton and the nucleus.

What are the three tiers of the MAPK cascade and their kinases?

1. MAPKKK → Raf (activated by GTP-Ras)
2. MAPKK → MEK (phosphorylated by Raf on two Ser residues)
3. MAPK → ERK (phosphorylated by MEK on one Thr and one Tyr residue)

Anki cloze

MEK is a {1:Ser/Thr and Tyr} protein kinase that phosphorylates ERK on a {2:Thr} and a {3:Tyr} residue, activating it.

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7. Nuclear Transcription Factors – Downstream of ERK

Nuclear Translocation of ERK

P-MAPKs like ERK move into the nucleus and phosphorylate several nuclear transcription factors (e.g., Fos, Jun, Myc, Elk1), activating them → stimulate the transcription and translation of a set of genes needed for cell division.

Leucine Zipper Domain (c-Fos:c-Jun Heterodimer)

In the “Leucine zipper” domain (gray), the hydrophobic residues of c-Fos and the hydrophobic residues of c-Jun pack together at the interface of the coiled-coil (leucines colored blue, other hydrophobic residues yellow). Residues from the “basic region” (purple) directly interact with the DNA (red).

What happens after ERK is activated in the MAPK cascade?

Activated (phosphorylated) ERK translocates into the nucleus and phosphorylates nuclear transcription factors such as Fos, Jun, Myc, and Elk1, activating them to stimulate transcription and translation of genes needed for cell division.

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8. KSR – Kinase Suppressor of Ras

Naming Caveat

“While Kinase Suppressor of Ras may be its name, phosphorylation may not be its game” — inactivating mutations in KSR suppress the phenotypic effects induced by activated Ras.

KSR Structure and Classification

• Members of the KSR family are close relatives of the Raf kinases.
• However, KSRs do not have an RBD (Ras-binding domain).
• The mammalian KSR proteins were found to lack a lysine residue normally required for the phosphotransfer reaction → question of whether KSRs possess intrinsic kinase activity remains debated.

KSR as a Scaffold Protein

KSR proteins have scaffolding activities:

• In quiescent cells: KSR is found in a multiprotein complex containing MEK; 14-3-3 sequesters the inactive KSR complex in the cytosol.
• On signal activation: KSR rapidly translocates to the plasma membrane to increase the local pool of MEK, facilitating MEK activation.
• KSR proteins can form side-to-side dimers with the Rafs → can function as an allosteric activator of Raf.
• KSR proteins contain FxFP docking sites for activated ERK → phosphorylation of KSR and the Rafs at S/TP sites → these feedback sites disrupt the signaling complexes and promote the release of KSR and the Rafs from the cell surface.

KSR Function Summary

KSR modulates the dynamics of Ras pathway signaling by both potentiating and attenuating Raf activity and signal transmission to MEK and ERK.

KSR1 Scaffold Complex Dynamics (McKay et al., 2009)

Figure 5 – KSR1 regulates the intensity and duration of ERK cascade activation

• (A) Quiescent cells: KSR1 prevents improper ERK cascade activation by sequestering MEK away from Raf.
• (B) Signaling cells – early: KSR1 first potentiates signal transmission from Raf to MEK by facilitating the Raf/MEK interaction.
• (C–D) Signaling cells – late: KSR1 then attenuates signaling by docking activated ERK, facilitating disruption of the KSR1 scaffold complex via feedback phosphorylation.

What is the dual role of KSR1 in ERK cascade signaling?

KSR1 first potentiates the signal by facilitating Raf/MEK interaction (increasing MEK availability at the membrane), and then attenuates the signal by docking activated ERK, which leads to feedback phosphorylation and disruption of the scaffold complex.

Anki cloze

In quiescent cells, KSR1 prevents improper ERK activation by {1:sequestering MEK away from Raf}. On activation, KSR1 first {2:potentiates} signal transmission, then {3:attenuates} it via feedback phosphorylation by activated ERK.

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9. Signal Termination – Rapid Deactivation

Rapid Re-establishment of Basal Conditions

Phosphorylation reactions and the Ras activation process stimulated by RTKs on the inner surface of cell membranes are very rapid events because:

• Specific tyrosine phosphatases (PTP)
• The intrinsic GTPase activity of Ras (further enhanced by GAP)

…re-establish the basic conditions.

Physiological Significance

Pathways emanating from Receptor Tyr-Kinases are involved in the regulation of gene expression and in the control of cell growth (“mitogenesis”) and differentiation.

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10. The Insulin Receptor (IR) – Detailed Mechanism

10.1 Structure

Insulin Receptor Structure

• Transmembrane glycoprotein of the RTK family
• Comprises 2 $\alpha$- and 2 $\beta$-subunits (${\alpha }_2{\beta }_2$) intertwined to form the insulin-binding site in the extracellular portion
• X-ray crystallography: the disulfide-bonded ectodomain of IR has a Λ-shaped structure
• Insulin does NOT enter cells, but binds to the $\alpha$-subunits and initiates a signal that travels a branched pathway (to the nucleus AND to insulin-sensitive enzymes in the cytosol)

What is the quaternary structure of the insulin receptor?

The insulin receptor is an ${\alpha }_2{\beta }_2$ heterotetramer. The two $\alpha$-subunits are extracellular (insulin-binding) and the two $\beta$-subunits are transmembrane with intracellular tyrosine kinase domains.

10.2 Signal Transduction Steps

Sequential Steps of Insulin Signaling

1. Insulin binds to $\alpha$-subunits → receptor undergoes autophosphorylation on carboxyl-terminal Tyr residues (the two juxtaposed $\beta$-chains phosphorylate each other)
2. Insulin receptor phosphorylates IRS-1 (Insulin Receptor Substrate-1) on its Tyr residues
3. SH2 domain of Grb2 binds to (P)-Tyr of IRS-1; Sos binds to Grb2, then to Ras → causing GDP release and GTP binding to Ras
4. Activated Ras binds and activates Raf-1
5. Raf-1 phosphorylates MEK on two Ser residues → MEK phosphorylates ERK on a Thr and a Tyr residue
6. ERK moves into the nucleus → phosphorylates nuclear transcription factors such as Elk1
7. Phosphorylated Elk1 joins SRF to stimulate the transcription and translation of a set of genes needed for cell proliferation (e.g., hexokinase, phosphofructokinase, pyruvate kinase)

Three Best Studied Nodes of Insulin Signaling

• Insulin receptor / IRS complex
• PI3 kinase (PI3K)
• AKT/PKB

Anki cloze

Once phosphorylated on several Tyr residues, IRS-1 becomes the point of {1:nucleation} for a complex of proteins including {2:GRB2-SOS-Ras-Raf} and {3:PI3K}.

10.3 Time Course of Insulin Effects

Immediate vs. Delayed Responses

Immediate responses (seconds/minutes after insulin binding):

• Increased glucose transport within adipocytes and skeletal muscle fibers
• Modification of the enzymatic activity and/or phosphorylation status of pre-existing proteins

Delayed transcriptional effects require several hours or days after initiation of insulin signaling (since insulin affects mechanisms of gene transcription).

Why does insulin have both immediate and delayed biological effects?

Immediate effects (seconds–minutes) result from direct modification of pre-existing proteins (e.g., increased glucose transport via GLUT4 translocation in adipocytes and muscle). Delayed effects (hours–days) result from transcriptional changes requiring new protein synthesis.

10.4 Additional Signaling Pathways Activated by Insulin

Grb2 is not the only protein that associates with phosphorylated IRS-1

Activation of additional signaling pathways leads to:

• ↑ Glucose uptake
• ↑ Glycogen biosynthesis
• ↑ Lipid biosynthesis
• ↓ Gluconeogenesis
• ↓ Glycogenolysis
• ↓ Lipolysis

List the metabolic effects of insulin signaling through IRS-1.

Activated: glucose uptake, glycogen biosynthesis, lipid biosynthesis. Inhibited: gluconeogenesis, glycogenolysis, lipolysis.

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🧠 Mnemonics

Mnemonic: MAPK Cascade Order

“Rabbits Make Every Kid Happy” → Raf → MEK → ERK → Kinase cascade → (gene expression for) Hyperproliferation

Mnemonic: Insulin metabolic effects (ON vs. OFF)

Insulin turns ON: Glucose uptake, Glycogen synthesis, Lipid synthesis → “Good GL”
Insulin turns OFF: Gluconeogenesis, Glycogenolysis, Lipolysis → “Bad GL” (catabolic)

Mnemonic: RTK activation steps

“DITAK” → Dimerization → Intracellular kinase domains contact → Trans-autophosphorylation → Adapter proteins (SH2 binding) → Kinase cascade

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TLDR

Lecture A.05 – Complete Summary

Insulin structure:

• Secreted as hexameric ${\text{Zn}}^{2+}$-insulin; must dissociate to monomer (aided by albumin) to bind receptor
• Dimer dissociation (loss of $\beta$-sheet, hydrophobic core solvation) is required for receptor binding

RTK overview:

• RTKs are single-pass transmembrane receptors with an intracellular tyrosine kinase domain (inactive without ligand)
• Ligand binding induces dimerization → trans/auto-phosphorylation of Tyr residues → signal persists even after hormone dissociation
• Deactivated by: (1) PTP-mediated Tyr dephosphorylation; (2) cAMP-driven Ser/Thr phosphorylation

Transducers and SH2 domains:

• Phospho-Tyr residues recruit SH2-domain-containing transducers (e.g., Grb2, Shc, PLC-γ, PI3K, c-Src, GAP)
• Grb2–SOS complex: SOS is a GEF that activates Ras by promoting GDP→GTP exchange

Ras cycle:

• GTP-Ras = active; terminated by intrinsic GTPase activity (enhanced by GAP)
• Oncogenic Ras: GTPase activity blocked → permanently active → drives cell proliferation

MAPK cascade (Ras → Raf → MEK → ERK):

• Raf (MAPKKK): activated by GTP-Ras; Raf-1 requires PP2A-mediated dephosphorylation + PAK/Src phosphorylation
• MEK (MAPKK): Ser/Thr+Tyr kinase, phosphorylated by Raf
• ERK (MAPK): phosphorylated by MEK on Thr+Tyr; translocates to nucleus
• Nuclear targets: Fos, Jun, Myc, Elk1 → cell division gene programs
• Entire cascade is scaffolded

KSR (Kinase Suppressor of Ras):

• Raf kinase relative, lacks RBD, debated intrinsic kinase activity
• Acts as scaffold: sequesters MEK in cytosol in quiescent cells (with 14-3-3); translocates to membrane on activation
• Potentiates signaling (facilitates Raf/MEK interaction), then attenuates (docks activated ERK → feedback phosphorylation → complex disruption)
• Can form side-to-side dimers with Rafs → allosteric activation of Raf

Insulin receptor (IR):

• ${\alpha }_2{\beta }_2$ transmembrane glycoprotein; Λ-shaped ectodomain; insulin binds $\alpha$-subunits (does NOT enter cells)
• Trans-autophosphorylation of $\beta$-chains → phosphorylation of IRS-1 → nucleation of GRB2-SOS-Ras-Raf + PI3K complexes
• Three key nodes: IR/IRS, PI3K, AKT/PKB
• Immediate effects (seconds–min): ↑ glucose transport in adipocytes and skeletal muscle; enzyme phosphorylation
• Delayed effects (hours–days): transcriptional activation of metabolic genes (hexokinase, PFK, pyruvate kinase)
• Metabolic outcomes: ↑ glucose uptake/glycogen/lipid synthesis; ↓ gluconeogenesis/glycogenolysis/lipolysis