Biologi Sel: Jalur Sinyal Utama

Major Signaling Pathways

• JAK/STAT pathway
• Ras/MAPK pathway
• PI3K/Akt pathway

Cytokine Signaling through the JAK/STAT Pathway

• Cytokines are small proteins (5-25 kDa)
• Released by cells to enable cell-cell communication
• Do not cross cell membranes
• Signal through binding to specific receptors on the cell surface
• Cytokine families include: Interleukins (IL), Interferons (IFN), and Tumor Necrosis Factor (TNF) family, Transforming growth factor (TGF)

Cytokine Receptors

• Hetero-dimers or trimers
• Crucial for immune regulation
• Different cytokines are produced by different cell types (e.g., IL-2 by T cells and DCs)
• Different receptors bind different cytokines and activate different JAKs and STATs

JAK/STAT Signaling

• JAKs (Janus kinases) are kinases
• Four members: JAK1, JAK2, JAK3, and TYK2
• Two kinase domains (JH1/JH2)
• JH2 is the autoinhibitory domain
• SH2 domain allows interaction with cytokine receptors
• FERM domain
• Receptors are not stable at the membrane without JAKs

Cytokine Signaling through the JAK-STAT Pathway (Mechanism)

• Cytokine-mediated receptor dimerization
• JAK tyrosine residues are phosphorylated
• STATS recruitment and JAK-mediated phosphorylation
• STATS dimerization
• Transcription/mRNA translation
• Transcription/ translation in cytokine-responsive gene

Signal Transducer and Activator of Transcription (STAT)

• Nuclear localization signal (NLS)
• Tyrosine (Y) and serine (S) residues are phosphorylated in response to extracellular stimuli
• Interaction sites for various transcriptional co-activators (green) and co-repressors (red)

STATs bind to DNA to promote gene transcription

• DNA sequences (e.g., CRE, ISRE, GASs) are found in the promoters of regulated genes and are involved in cytokine response and interferon (Type I, II, and III)
• Specific sequences are associated with particular type of interferons

IFNs activate unique gene transcription

• Different types of interferons utilize different JAK/STAT combinations to activate unique gene transcription and promote immune functions
• Type I IFNs use IFNAR1 and IFNAR2 receptors with specific JAK and STAT combinations
• Type II IFNs use IFNGR2 receptor with specific JAK and STAT combinations
• Type III IFNs use IFNLR1 and IL-10RB receptor with specific JAK and STAT combinations

Different cytokine receptors can activate different JAKs and STATS

• Different cytokine receptors activate specific combinations of JAKs and STATs, leading to diverse downstream signaling pathways.

Mutations in JAK2

• JAK2 mutations (e.g., V617F) are associated with myeloproliferative neoplasms
• Destabilizes the interaction between JH2 and JH1 domains
• Results in overproduction of myeloid progenitor cells
• Ruxolitinib (Jakafi) is a potent inhibitor

Structure and activation of JAK2

• Mutation allows for partial activation
• Cytokines activation result full activation of the kinase (e.g., erythropoietin, thrombopoietin)
• These cytokines promote blood development

Turning JAK/STAT Signaling Off

• PIAS (protein inhibitor of activated STAT) inhibits DNA binding
• SOCS (suppressor of cytokine signaling) blocks STAT recruitment and inhibits JAK
• Protein tyrosine phosphatases (PTPs) dephosphorylate JAK or STAT receptors

Ubiquitin degradation pathway

• Ubiquitin marks proteins for degradation in the proteasome
• A multi-step pathway involving E1, E2, and E3 ubiquitin ligases

Ras/MAPK Pathway

• Ras is a small GTPase involved in cellular signaling
• Molecular switch similar to Ga subunit in heterotrimeric G-protein
• Most common oncogene
• Three common types of Ras: HRas, KRas, and NRAS
• Lipid modification critical for activity
• MAPK is mitogen-activated protein kinases
• Mitogen is a factor that stimulates cell growth

Activation of Ras by Growth Factor Receptors

• Tyrosine Kinase Receptors act as dimers to trigger trans-phosphorylation
• Upon activation, a conformational change occurs
• Examples include: Insulin receptor, epidermal growth factor, and platelet-derived growth factors

Insulin Receptor Signaling Overview

• Insulin binds to the insulin receptor, causing autophosphorylation
• Receptor then phosphorylates IRS1
• IRS1 activates Grb2, which activates Sos
• Sos activates Ras, which activates Raf-1, causing a cascade
• The activation of ERK occurs leading to the transcription of genes for cell division

Adapter proteins

• IRS1: insulin receptor substrate 1, recruits Grb1 when phosphorylated
• Grb2: growth factor receptor bound 2, links IRS1 with Ras GEF Sos, binds Sos through SH3 domain

Sos

• Major Ras GEF
• Drosophila orthologue of "son-of-sevenless"
• Regulates development of 7th central photoreceptor in Drosophila (detects UV light)
• Receptor tyrosine kinase

Activation of Ras

• Sos causes GTP release from Ras, activating Ras
• Ras activates Raf-1, a serine/threonine kinase
• Raf-1 activates MEK
• MEK activates ERK

Activation of the MAPK cascade

• Raf activates MEK
• MEK activates ERK, with each step involve a phosphorylation
• ERK moves to the nucleus
• Phosphorylates key transcription factors

Scaffold proteins keep MAPK modules together

• Some proteins cluster some MAPK modules together for efficiency
• Too many or few scaffold proteins can lead to decreased signal efficiency

ERK activates transcription factors

• ERK moves to the nucleus and phosphorylates key transcription factors
• Transcription factors promote transcription of genes required for cell growth
• Examples of Transcription factors: c-Fos, and c-Jun

Other MAPK pathways

• JNK (Stress-activated c-Jun N-terminal kinase) : Stress activated
• p38: Stress activated and tends to promote apoptosis

Generation of the AP-1 transcription factor

• ERK and JNK phosphorylate c-Jun and c-Fos.
• c-Fos and c-Jun dimerize to form AP-1
• AP-1 is a transcription factor

The same receptor can activate all or some of the MAPK pathways

• The balance of signals dictates the fate of the cell (e.g., proliferation or apoptosis)

Oncogenic Ras

• Ras mutations occur in ~30% of human cancers
• Single-base pair mutations generate amino acid changes
• Blocks GTPase activity (resulting in constitutively activated Ras)
• Untargetable

Example Ras mutations

• Specific mutations in different codons (12,13,61) for each HRAS, KRAS, NRAS.
• Specific counts of these mutation for each cancer that related to.

Oncogenic B-Raf mutations

• V600E (class I) is the most common mutation in oncogenic B-Raf mutations
• Causes kinase to be constitutively active
• Particular common in melanoma and lung
• Targetable with small molecules (e.g.,Vemurafenib, Dabrafenib)

Phosphatidylinositol-3-kinase (PI3K) signaling overview

• PI3K phosphorylates PIP2 to PIP3
• PI3K pathway is activated by growth factors.
• PI3k pathway affects mRNA translation, lipid synthesis, Nucleotide synthesis, and Autophagy

PI3K: a family of proteins

• Phosphorylates the 3’ position of phosphatidylinositols
• Creates different phosphorylated products: (PtdIns4P, PtdIns(3,4)P2, PtdIns(4,5)P2, and PtdIns (3,4,5)P3).

Class I PI3K have two subunits

• p110 (catalytic subunit)
• p85 (regulatory subunit)
• p85 binds to Gβγ or phosphorylated receptors
• Recruits p110 to the membrane
• Phosphorylates PI(4,5)P2

It's the PIP3 that is key for the next steps

• PIP3 can bind to proteins with PH domains to recruit them to the plasma membrane, enabling activation
• Akt/Protein Kinase B is a major downstream mediator of PI3K
• Akt is recruited to the plasma membrane via its PH domain
• Akt is phosphorylated by PDK1 and mTORC2
• This double phosphorylation activates Akt

Akt/PKB activation

• Akt activation requires phosphorylation on Thr308 (by PDK1) and Ser473 (by mTORC2).
• PDK1 and mTORC2 both have PH domains

mTORC

• TOR: Target of Rapamycin
• mTOR: mammalian target of Rapamycin
• mTORC: mammalian target of Rapamycin complex
• Rapamycin: antifungal metabolite from Streptomyces hygroscopicus, potent immunosuppressant and anti-proliferative, specifically targets mTORC1.

mTORC1(Raptor) and mTORC2 (Rictor)

• Different mTORC1 and mTORC2 involve in protein synthesis, nucleotide synthesis, lipid synthesis, autophagy, glycolysis, actin/cytoskeleton organization pathways

mTORC1 activation

• Growth factors activate mTORC1 to promote proliferation
• mTORC1 is associated with insulin, glucose, and amino acids

AMPK (AMP Kinase) inhibits growth

• AMPK inhibits growth when AMP levels are high, affecting glucose, amino acids, and growth factors signaling
• AMP increased when cells do not have enough energy
• Regulates cell cycle, survival, and processes

Wnt signaling

• WNT is a secreted protein from Drosophila's Wingless (Wg) gene
• Directs the fate of cells
• Directs patterning in central nervous system, respiratory and circulatory systems, and more epidermal structures

Secreted protein (Wnt)

• Wg does not act in a cell autonomously
• In heterozygous flies, some rescues occur, due to the presence of normal protein rescuing neighboring cells

Discovery of the int-1 oncogene

• MMTV insertion mutation to identify oncogenes
• MMTV promoter drives high expression of adjacent host genes
• Int-1, a gene associated with the formation of mammary tumor
• Int-1 has 54% similarity with Wg (Wingless)
• Renamed Wnt

Normal Wnt function

• Found in the nervous system of embryos
• KO mutations in mice eliminate brain patterning
• Overexpression in Xenopus causes duplication of the frog embryo's axis
• Wnt acts as an indicator to regulate brain patterning

Gradient of Wnt allows anterior/posterior development

• Wnt gradient affects development of anterior and posterior structures
• Wnt/b-catenin signaling and retinoic acid level gradient affect cell type (anterior and posterior)
• Wnt/b-catenin high/retinoic acid low to Wnt/b-catenin low/retinoic acid high

Overview of Wnt signaling

• Axin, APC, CK1α, GSK-3β form a destruction complex, important for the regulation of Wnt signaling
• GSK-3β phosphorylates Ser33, Ser37, and Thr41 of β-catenin
• Frizzled: receptor for Wnt
• LRP: Low-density lipoprotein receptor-related protein
• GSK-3β: glucogen synthase kinase-β
• CK1α: Casein Kinase a
• APC: Adenomatous Polyposis Coli

Overview of Wnt signaling

• Wnt binds LRP and Frizzled.
• Dishevelled recruits the destruction complex to LRP to prevent degradation of β-catenin
• Free β-catenin translocates to the nucleus to activate transcription factors (e.g., TCF/LEF) for gene expression

GSK-3β

• Phosphorylation inhibits GSK-3β activity
• Serine/threonine kinase
• Involved in inactivating glycogen synthase in insulin signaling
• Involved in other signaling pathways like NF-kB, Hedgehog, and Notch

Mutations in Wnt signaling

• APC (Adenomatous Polyposis Coli) protein loss-of-function mutations are commonly associated with starting events of sporadic colorectal cancer
• Familial adenomatous polyposis coli (FAP): inherited predisposition for cancer

β-catenin

• Two pools of β-catenin (cytoplasmic & membrane bound)
• Cytoplasmic β-catenin is associated with GSK-3β, APC, and Axin complex
• Membrane-bound β-catenin is associated with E-cadherin
• Important for cell-cell adhesion
• Phosphorylation from RTK can lead to activation

β-catenin in cancer

• Mutations in β-catenin can resist to degradation
• Disrupt phosphorylation that induces degradation (e.g., Ser33, Ser37, and Thr41
• Nuclear β-catenin accumulation correlate with colorectal cancer development
• Cooperates with loss of p53 to promote carcinogenesis

Notch signaling

• Notch is a juxtacrine signaling pathway
• Discovered in Drosophila in the 1910s
• In mammals, Notch prevents differentiation of cells into neurons

Multiple Notch and Delta family genes

• Notch family genes (NOTCH1, NOTCH2, NOTCH3, NOTCH4)
• Delta family genes (e.g., DLL1, DLL3, DLL4, JAG1, JAG2)

Notch signaling overview

• Activated Notch is cleaved into different pieces that associate at the membrane, releasing the cytoplasmic domain which translocates to the nucleus and activates gene transcription

Regulation of S2 cleavage

• The S2 cleavage site is normally hidden.
• Upon binding to a ligand, a conformational change occurs, exposing the cleavage site.
• Cleavage of the protein is performed by ADAMs enzymes (ADAM 10, ADAM 17, and ADAMTS1), which forms the NOTCH extracellular truncation (NEXT)

The S3 cleavage

• y-secretase cleaves Notch
• The substrates are NOTCH receptors and amyloid precursor protein
• The mechanism of y-secretase cleavage is not clearly understood.

Other functions of Notch signaling

• Notch signaling promotes organ production and damage repair
• Affects cells differentiation
• Stimulated in response to injuries

Disease associated with Notch

• Alagille syndrome: abnormal development of liver, heart, vasculature, bones, eyes, and maxillofacial dysplasia; chronic cholestasis in children; severe liver damage
• Notch signaling is required for regeneration of liver cells