Major Signaling Pathways (JAK/STAT, Ras/MAPK, PI3K/Akt) PDF

Major signaling pathways 1. JAK/STAT 2. Ras/MAPK 3. PI3K/Akt 1. Many cytokines signal through the JAK/STAT pathway Cytokines 5-25 kDa proteins (ie small) Released by cells to enable cell-cell communication Do not cross cell membranes Signal via binding...

Major signaling pathways 1. JAK/STAT 2. Ras/MAPK 3. PI3K/Akt 1. Many cytokines signal through the JAK/STAT pathway Cytokines 5-25 kDa proteins (ie small) Released by cells to enable cell-cell communication Do not cross cell membranes Signal via binding to specific receptors on the cell surface Cytokine families Interleukins (IL)- JAK/STAT Interferons (IFN)- JAK/STAT Tumor Necrosis Family (TNF) Transforming growth factor (TGF) Cytokine Receptors - Hetero-dimers or -trimers - Particularly important in immune regulation JAK/STAT signaling JAK: Just Another Kinase/ Janus Kinase 4 members: JAK1, JAK2, JAK3, and TYK2, 2 domains that look like kinase domains (JH1/JH2) JH2 is the autoinhibitory domain SH2 domain Allow interaction with cytokine receptors, without JAKs, receptors FERM domain are not stable at the membrane Signal Transducer and activator of transcription (STAT) Nuclear localization signal (NLS) Tyrosine (Y) and serine (S) residues phosphorylated in response to extracellular stimuli sites of interaction of various transcriptional co- activators (green) and co- repressors (red) https://doi.org/10.1186/s13045-021-01214-y STATs bind to DNA to promote gene transcription DNA sequences are found in the promoters of regulated genes Cytokine response element (CRE)- general Interferon-sensitive response element (ISRE) – Type I and III IFN specific Gamma-activated sequences (GASs)- Type II IFN specific IFNs activate unique gene transcription to promote immune functions by using unique JAK/STAT combinations ISGF3 https://doi.org/10.1038/s12276-021-00592-0 Different cytokine receptors can activate different JAKs and STATs  Mutations in JAK2 are associated with myeloproliferative neoplasms (cancer) Cancers of blood cells (myeloid cells) V617F mutation Destabilizes the interaction between the JH2 and JH1 domains Results in overproduction of myeloid progenitor cells Inhibitor: Ruxolitinib (Jakafi) competitively inhibits the ATP-binding catalytic site on JAK1 and JAK2  Structure and activation of JAK22 Arrows indicate the positions of the most frequently mutated regions Mutation allows kinase to be partially active Addition of a cytokine (eg erythropoietin or thrombopoietin) fully activates the kinase These cytokines promote blood cell development https://doi.org/10.1016/j.exphem.2015.06.007  Turning JAK/STAT signaling off 1. PIAS (protein inhibitor of activated STAT) Inhibits DNA binding 2. SOCS (suppressor of cytokine signaling) Blocks STAT recruitment Inhibits JAK Degrades JAK & STAT 3. PTP (protein tyrosine phosphatases) Dephosphorylate the receptor, JAK or STAT https://doi.org/10.1038/s41392-021-00791-1  Ubiquitin degradation pathway https://doi.org/10.1038/s41392-022-00966-4 2. Ras/MAPK Ras: a small molecular weight G protein A molecular switch (like the G𝛼 subunit of heterotrimeric G proteins) Most common oncogene H-Ras, K-Ras, N-Ras Lipid modification critical for activity MAPK= mitogen-activated protein kinases Mitogen is a factor that stimulates cell growth https://doi.org/10.1038/nrc969 Activation of Ras by Growth Factor Receptors Tyrosine Kinase Receptors Dimers Trans-phosphorylate upon activation due to a conformational change Examples: insulin receptor (INSR), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) Insulin Receptor signaling overview Adapter proteins IRS1 Insulin receptor substrate 1 When phosphorylated, IRS1 recruits Grb1 Grb2 Growth factor receptor bound 2 Links IRS1 with the Ras GEF, Sos Binds Sos through its SH3 domain  Sos WT SEV-/- Major Ras GEF “son-of-sevenless” Drosophila orthologue “Sevenless” gene in Drosophila regulates the development of the 7th central photoreceptor (detects UV light) in each ommatidium Receptor tyrosine kinase Sos is downstream of the “sevenless” gene so it was called “son-of-sevenless” 7 6 rhabdomeres rhabdomeres https://doi.org/10.1016/S0167-4889(00)00020-3 Activation of Ras Sos causes release of GDP from Ras GTP binds and Ras is activated GTP-Ras binds and activates Raf-1 Raf-1: a serine/threonine kinase Activation of the MAPK cascade (Ser/Thr kinases) Raf MAPKKK MAPK kinase kinase P P MEK MAPKK MAPK kinase P P ERK MAPK  Scaffold proteins keep MAPK modules together Inactive Active Too many scaffold proteins or too few scaffold proteins will decrease signal efficiency KSR: kinase suppressor of Ras https://doi.org/10.3389/fphys.2012.00475 ERK activates transcription factors ERK moves to the nucleus Phosphorylates key transcription factors Transcription factors promote transcription of genes required for cell growth Other MAPK pathways JNK Stress activated c-Jun N-terminal kinase p38 Stress activated Tends to promote apoptosis Generation of the AP-1 transcription factor Hetero- or homo-dimers of Jun, Fos, ATF, or Maf basic-leucine zipper DNA-binding proteins c-Jun and c-Fos are immediate-early response genes oncogenic The same receptor can activate all or some of the MAPK pathways- the balance of signals will dictate the fate of the cell (eg proliferation vs apoptosis) Oncogenic Ras Mutation occurs in ~30% of all human cancers Single base pair mutation creates a single amino acid mutation (codons 12, 13, 61) Blocks Ras GTPase activity so Ras is constitutively on Currently untargetable https://doi.org/10.1158/0008-5472.CAN-11-2612 Example Ras mutations https://doi.org/10.1158/0008-5472.CAN-11-2612 Oncogenic B-Raf mutations V600E (class I) is the most common mutation Causes kinase to be constitutively active Particularly common in melanoma and lung Mutated in ~7% of cancers Targetable with small molecules Vemurafenib Dabrafenib doi: 10.3390/genes11111342 3. Phosphatidylinositol-3-kinase (PI3K) signaling overview  PI3K: a family of proteins that phosphorylate the 3’ position of phosphatidylinositides https://doi.org/10.3390/ijms19123931  Class I PI3K have 2 subunits p110: catalytic subunit p85: regulatory subunit p85 binds to Gβ𝛾 or phosphorylated receptors Recruits p110 to membrane Phosphorylates PI(4,5)P2 https://doi.org/10.3390/ijms19123931  It’s the PIP3 that is key for the next steps PIP3 can be bound by proteins with PH domains to recruit them to the plasma membrane and allow them to be activated Akt/Protein Kinase B is a major downstream mediator of PI3K It is recruited to the plasma membrane via its PH domain Then gets phosphorylated by PDK1 and mTORC2 This double phosphorylation activates AKT  Akt/PKB activation Full activation requires phosphorylation on Thr308 (PDK1) and Ser473 (mTORC2) PDK1 also has a PH domain One of the components of mTORC2 (mSin1) also has a PH domain https://doi.org/10.1038/nri2888  Akt/PKB activation mTORC2 phosphorylates Ser473 which removes the autoinhibitory interaction between the PH domain and the kinase domain PDK1 phosphorylates the kinase domain activation loop at Thr308 to activate the kinase https://doi.org/10.7554/eLife.59151 mTORC TOR: target of Rapamycin mTOR: mammalian target of Rapamycin mTORC: mammalian target of Rapamycin complex Rapamycin: antifungal metabolite produced by Streptomyces hygroscopicus Potent immunosuppressive and anti-proliferative activities Specifically targets mTORC1  mTORC1(Raptor) and mTORC2 (Rictor) doi: 10.1016/j.cmet.2014.01.001  mTORC1 activation Growth Factors activate mTORC1 to promote proliferation  mTORC1 activation Growth Factors activate mTORC1 to promote proliferation  mTORC1 activation Amino acids (nutrients) activate mTORC1 to promote proliferation  mTORC1 activation AMPK (AMP Kinase) inhibits growth when AMP levels are high  mTORC1 activation When glucose (nutrients) increases ATP levels, AMPK is inhibited which allows mTORC1 to be activated promote proliferation  A bit on the TSC1/2 complex TSC1/2 acts as a GAP to generate Rheb-GDP TSC1/2 is inhibited by phosphorylation Allows Rheb-GTP to accumulate Rheb-GTP activates mTORC1 https://doi.org/10.1038/cr.2007.106  mTORC2 Dual specificity kinase: phosphorylates Ser/Thr and Tyr Less is known about it compared to mTORC1 Regulates the actin cytoskeleton Work from my lab identified a role for mTORC2 in the formation of bioactive extracellular vesicles  Mutations that activate PI3K signaling are highly prevalent in cancer PTEN (Phosphatase and TENsin homolog deleted on chromosome 10) Tumor suppressor Removes phosphate from PIP3 Also dephosphorylates downstream targets of Akt Truncating mutations (positions 233, 267, and 319–323) Deletions https://doi.org/10.3390/ijms19123931  Common PTEN mutations doi: 10.3390/genes11070719  PI3K pathway inhibitors in clinical trials https://doi.org/10.1186/s12943-019-0954-x  PI3K pathway inhibitors in clinical trials https://doi.org/10.1186/s12943-019-0954-x Ras can activate PI3K Wnt signalling Wnt Comes from the Drosophila gene- Wingless (Wg) Secreted protein Directs cell fate and morphogenesis patterning the central nervous system, the gut, the respiratory and circulatory systems, and various epidermal structures https://europepmc.org/article/med/29618590 Secreted protein Wg does not act cell autonomously In heterozygous flies, get some rescues because the presence of the normal protein rescues the neighbouring cells Wg antibody staining (red) shows that the protein is expressed in stripes in WT embryos, and the protein is detected over several cell diameters on either side of the stripe https://europepmc.org/article/med/29618590 Discovery of the int-1 oncogene Used integration mutants to identify oncogenes MMTV (mouse mammary tumor virus) DNA was randomly inserted into the genome of cells The strong MMTV promote drives high expression of adjacent host genes Called the genes that caused mammary tumor formation : int int-1 was the first gene identified Had 54% identify with Wg New gene name: Wnt (Wg + int) Normal Wnt function Found in the nervous system of embryos KO mutations in mice eliminate patterning of the brain Overexpression in Xenopus cause duplication of the frog embryo’s axis (closed arrows; normal axis open arrows) https://www.cell.com/cell/pdf/0092-8674(89)90506-0.pdf Gradient of Wnt allows anterior/posterior development https://doi.org/10.1016/j.stemcr.2020.08.016 Overview of Wnt signaling Normally, the pathway is maintained in an inactive state Axin, APC, CK1𝛼, GSK-3β form destruction Degradation complex via the GSK-3β phosphorylates Ser33, Ser37, and proteosome Thr41 to promote degradation of β-catenin Frizzled: receptor for Wnt LRP: LDL-receptor-related protein GSK-3β: glucogen synthase kinase-β CKI𝛼: Casein Kinase 𝛼 APC: Adenomatous Polyposis Coli https://archive.org/details/alberts-molecular-biology-of-the-cell-7th/page/931/mode/2up?q=wnt  Overview of Wnt signaling Wnt binds LRP and Frizzled Disheveled recruits the destruction complex to LRP to prevent degradation of β-catenin β-catenin translocates and accumulates in the nucleus β-catenin binds TCF/LEF transcription factors to promote gene transcription https://archive.org/details/alberts-molecular-biology-of-the-cell-7th/page/931/mode/2up?q=wnt GSK-3β Phosphorylation can inhibit its activity Serine/threonine kinase Initially identified for its role in inactivating glycogen synthase in insulin signaling (more on this later in the course) Also involved in NF-𝜅B, Hedgehog, and Notch signaling (more on some Sites of Phosphorylation of GSK-3beta of these later) which Regulate its Activity. https://doi.org/10.18632/oncotarget.2037  Mutations in Wnt signaling promote colorectal cancer APC (Adenomatous Polyposis Coli ) protein is a tumor suppressor Inactivating mutations are initiating events for ~80% of sporadic colorectal cancers Familial adenomatous polyposis coli (FAP): inherited predisposition to cancer  β-catenin Two pools of β-catenin Cytoplasmic: associated with GSK-3β, APC, Axin complex Membrane bound: associated with E-cadherin Important role in cell-cell adhesion Can add to the cytoplasmic pool if interaction with E-cadherin disrupted Can be phosphorylated by RTK to cause activation https://www.tandfonline.com/doi/full/10.1080/21655979.20 23.2251696#d1e442 β-catenin in cancer Mutations in β-catenin (CTNNB1) can make it resistant to degradation For example: Ser33, Ser37, Thr41 Disrupts phosphorylation that induces degradation Nuclear β-catenin accumulation correlates with colorectal cancer progression (eg. normal- 0%; polyps- 8%; carcinoma- 100% Cooperates with loss of p53 (tumor suppressor) to promote carcinogenesis https://www.tandfonline.com/doi/full /10.1080/21655979.2023.2251696#d 1e442  β-catenin untargetable? Focus on hotspots to block specific protein-protein interactions https://www.tandfonline.com/doi/full /10.1080/21655979.2023.2251696#d 1e442 Notch signalling Juxtacrine signaling Notch was discovered in Drosophila in 1910s Wings were ‘notched’ and not rounded when the gene was mutated Full knock-out was lethal  In mammals, Notch prevents differentiation of cells into neuronal cells by neighboring cells that are developing into neurons Which cell expresses notch and with expresses the ligand, delta, is a competition. Each cell expresses both at first, then one “wins” by expressing more delta. Or other factors can stabilize delta https://archive.org/details/alberts-molecular-biology-of-the-cell-7th/page/927/mode/2up?q=notch Multiple Notch and Delta family genes Notch family NOTCH1, NOTCH2, NOTCH3, and NOTCH4 Delta family DLL1 governs cell differentiation and cell-to-cell communication DLL3 suppresses cell growth by inducing apoptosis DLL4 activates NF-κΒ signaling to enhance vascular endothelial factor (VEGF) secretion and tumor metastasis JAG1 enhances angiogenesis JAG2 promotes cell survival and proliferation https://doi.org/10.1038/s41392-022-00934-y  Notch signaling overview After Notch translation, it is cleaved (S1) into two pieces that associate with each other at the membrane Upon binding to Delta, another cleavage (S2) removes the extracellular domain of Notch. This gets endocytosed by the Delta-expressing cell This cleavage releases the cytoplasmic domain A final cleavage (S3) releases the cytoplasmic tail of the activated Notch The tail translocates to the nucleus and activates gene transcription NICD: Notch intracellular domain NEXT: Notch extracellular truncation Regulation of S2 cleavage The S2 cleavage site is normally hidden Upon binding of ligand, a conformational change occurs and the cleavage site is exposed Protein is cleaved by ADAM 10, ADAM 17, and ADAMTS1 ADAM (a disintegrin and metalloprotease) Creates NOTCH extracellular truncation (NEXT) The S3 cleavage 𝛾-secretase Classical substrates are NOTCH receptors and amyloid precursor protein Mechanism not clear  Other functions of Notch signaling organ production and damage repair organ production and damage repair https://doi.org/10.1038/s41392-022-00934-y Disease associated with Notch Example: Alagille syndrome abnormal development of the liver, heart, vasculature, bones, eyes, and maxillofacial dysplasia chronic cholestasis in children Severe liver damage Notch is required to regenerate BEC cells (bile duct epithelial cells) from hepatocytes https://doi.org/10.1038/s41392-022-00934-y