Signaling via Kinases lecture 4-4 2024-2.pdf

Receptor tyrosine kinases (RTKs) Topics Receptor Tyrosine Kinases (RTKs) The Ras/MAP Kinase Pathway Phosphoinositide Signaling Goals Learn the properties of RTKs. Learn about RTK signaling via the Ras/MAP kinase signaling pathway. Learn the general features of the PI-3 kinase signaling pathway. Mode...

Receptor tyrosine kinases (RTKs) Topics Receptor Tyrosine Kinases (RTKs) The Ras/MAP Kinase Pathway Phosphoinositide Signaling Goals Learn the properties of RTKs. Learn about RTK signaling via the Ras/MAP kinase signaling pathway. Learn the general features of the PI-3 kinase signaling pathway. Models of RTK bias sgnaling. PDZ-PSD-95, disc large , Zona occuludens -1 Activation of RTKs via Ligand Binding Receptor tyrosine kinases (RTKs) regulate cell differentiation and proliferation. In some cases e.g., the epidermal growth factor (EGF) ligand binding causes receptor dimerization. In other cases (e.g., the insulin RTK), binding occurs to preexisting dimers. Binding of ligand Changes shape of ECD allowing Them to interact 1. Causes dimerization 2. Alters interaction of Intracellular domains How this occurs will be Discussed at end of lecture Activation of RTKs via Ligand Binding RTKs exhibit intrinsic tyrosine kinase activity located within their cytosolic domains that is inactivity in absence of ligand The binding of ligand activates the kinase domains which crossphosphorylate the two monomers of the dimeric receptor. Phosphorylation first occurs at a regulatory site known as the activation lip. Phosphorylation of the lip causes conformational changes that allow the kinase domain to phosphorylate other tyrosine residues in the receptor and in signal transduction proteins. Recruitment of Signal Transduction Proteins to Activated Receptors The amino acids surrounding each phosphorylated tyrosine provides a unique site for binding on receptor Signal transduction proteins interact with phosphorylated RTKs via phosphotyrosine binding domains. Insulin receptor substrate-1 (IRS-1). Binding of signaling proteins to IRS-1 allows them to be phosphorylated by the On signal transduction proteins-Two main binding domains-PTB and SH2 receptor (Src homology domain-2 Recruitment of Signal Transduction Proteins to Activated Receptors Example of transducter proteins: These include proteins such as GRB2/SOS involved in activation of the Ras GTPase. GB2 Binds RTK SOS GEF RAS MAP Kinase pathway Small G proteins Ras is a monomeric (small) GTPase switch protein that unlike trimeric G proteins does not directly bind to receptors. Ras typically relies on guanine nucleotide-exchange factors (GEFs) for binding GTP, and on GTPaseactivating proteins (GAPs) for stimulation of GTP hydrolysis. Once activated, Ras propagates signaling further inside the cell via a kinase cascade that culminates in the activation of members of the MAP kinase family. MAP kinases phosphorylate TFs that regulate genes involved in the cell cycle and in differentiation. RTK Activation of Ras The mechanism by which EGF activates Ras. Step 1, EGF binding causes receptor dimerization and autophosphorylation on cytosolic tyrosines. Step 2, the adaptor protein GRB2 binds receptor phosphotyrosine residues via its SH2 domain. GRB2 contains SH3 domains allow binding to GEF protein known as Sos Sos then recruits Ras to the complex. RTK Activation of Ras Step 3, In the last step of Ras activation , Sos promotes GTP exchange for GDP on Ras. The activated Ras-GTP complex then dissociates from Sos but remains tethered to the inner leaflet of the cytoplasmic membrane via a lipid anchor sequence. The active form of Ras then activates the MAP kinase portion of the signaling pathway Ras Activation of MAP Kinase Ras activates MAP kinase via a phosphorylation cascade that proceeds from Ras to Raf kinase, to MEK kinase, and finally to MAP kinase. MAP kinase then dimerizes and enters the nucleus MAP kinase=ERK MAP Kinase Activation of Transcription In the final steps of RTK-Ras/MAP kinase signaling, MAP kinase phosphorylates and activates the p90RSK kinase in the cytoplasm. Both kinases enter the nucleus where they phosphorylate ternary complex factor (TCF) and serum response factor (SRF), respectively. The phosphorylated forms of these TFs bind to serum response element (SRE) enhancer sequences that control genes such as c-fos. c-fos activates the expression of genes that propel cells through the cell cycle. SREs occur in a number of genes that are regulated by growth factors present in serum. constitutively GTP-bound, resulting in the activation of downstream effector pathways in the absence of extracellular stimuli (Karnoub and Weinberg, 2008). However, RAS GAPs have emerged as an expanding new class of tumor suppressor genes. Accumulating evidence indicates that inactivation of several RAS GAP family members represents an important and alternative mechanism to (hyper) activate RAS and its downstream effectors in tumors (Fig. 1). This review will focus on the emerging biological role of RAS GAPs in human cancer. There are 14 predicted RAS GAP genes in the human genome (Bernards, 2003). All contain a RAS GAP domain but share little similarity outside of this region (Fig. 2). Interestingly, members of the IQGAP subfamily contain an alternative amino acid within a key region of the catalytic domain, and therefore do not exhibit RAS GAP activity but still affect RAS signaling (Brill et al., 1996; Wang et al., 2007; Weissbach et al., 1994). Regions flanking the RAS GAP domains are thought to promote protein–protein and protein–lipid interactions, second messenger binding and phosphorylation by protein kinases. These interactions may facilitate association with specific subcellular membranes or compartments, regulating spatially restricted GTPase activation (Bernards and Settleman, 2004). Accordingly, individual RAS GAPs may target specific GTPases by associating with specific membranes and/or signaling complexes. Furthermore, RAS GAP activity and its effects on downstream signaling pathways appears to be regulated in response to specific growth factors which is likely tissue- and contextspecific (Bernards and Settleman, 2005; Cichowski et al., 2003; McGillicuddy et al., 2009). Finally, at least some RAS GAPs also have distinct RAS-independent functions (Min et al., 2010; White et al., 2012). Together, this illustrates that RAS GAPs play diverse and discrete, yet non-redundant, roles in cell signaling. The existence of distinct sets of modular domains in different RAS GAP proteins further indicates that RAS GAP activity is highly complex and very tightly regulated. Nevertheless, still Clinical relevance: Mutant RTKs and Ras/MAP kinase signaling proteins are associated with nearly all cancers. For example, Dominant Ras mutations that block GAP binding and lock Ras in the "on" state. RAS mutation occur in about 30% of human cancers and cause persistent activation of the Map Kinase pathway. Fig. 1. The RAS GTPase cycle and its deregulation in cancer. (A) RAS proteins together with their two key regulators, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), constitute molecular switches that cycle between ‘on’ and ‘off’ conformations caused by the binding of GTP or GDP, respectively. (B) Activating mutations in RAS genes occur in many different tumor types. These mutations typically render RAS constitutively GTP-bound, resulting in the activation of downstream effector pathways in the absence of extracellular stimuli. (C) Increasing evidence suggests that inactivation of several RAS GAP family members represents an important and alternative mechanism to (hyper)activate RAS and its downstream effectors in cancer. Clinical relevance: MEK Inhibitors Trametinib is a highly selective reversible allosteric inhibitor of MEK1 and MEK2 activity. Cobimetinib specifically binds to and inhibits the catalytic activity of MEK1, resulting in inhibition of extracellular signal-related kinase 2 (ERK2) phosphorylation Recruitment of many Signal Transduction Proteins occurs to activated Receptors Example of transducter proteins: Phosphatidylinositol-3 kinase (PI-3 kinase) Phospholipase Cg (PLCg) Both binding via SH2 domains. Signaling via IP3/DAG pathway. PLCg binds to activated RTKs via SH2 domains. RTKs also can signal via formation of phosphoinositide compounds. Like GPCRs, they signal via the IP3/DAG pathway. Signaling via Phosphatidylinositol 3-phosphates In addition, RTKs can signal via PI 3,4-bisphosphate and PI 3,4,5-trisphosphate formed by the enzyme PI-3 kinase. The SH-2 domain of PI-3 kinase binds to phosphorylated IRS-1 bound to the RTK The PI 3-phosphate compounds synthesized by PI-3 kinase activate protein kinase B (PKB). Activation of Protein Kinase B Signaling downstream of PI 3-phosphates is conducted by PKB. PKB is recruited to the membrane via binding to PI 3-phosphates via its PH domain (pleckstrin-homology). There it is phosphorylated and activated by the PDK1 & mTorc2 kinases. PDK1 also is recruited to the membrane via binding to PI 3phosphates. Activated PKB then enters the cytosol, where it phosphorylates target proteins PKB is also known as AKT mTORC2 PKB/AKT plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, proliferation, differentiation, transcription, and migration. PI 3-phosphate signaling ultimately is terminated by cleavage of 3-phosphates from phosphoinositides by the PTEN. Phosphatase and tensin homolog (PTEN) Figure 1 19 subfamilies of RTKs Diversity of the human RTK ligands. RTKs are listed on the top categorized into 19 different families as originally described and recently revised to remove the LMR1-3 family because of its reclassification as Ser/Thr receptor kinases. Ligands for each RTK family are shown underneath in depicted in mature, secreted form. The ephrin ligands remain membrane-embedded and typically signal in the juxtacrine fashion activating their cognate receptors on neighboring cells. The ligands are drawn with their N-terminus at the bottom. Main structural domains found in the RTK ligands are depicted in a cartoon form, according to the inserted legend, in their known oligomeric state except for angiotensins*, which may form higher-order oligomers in addition to dimers. Sizes of individual domains are not drawn up to scale. RTK, receptor tyrosine kinase. EGFR family Three of the four different EGFR/HER receptors, EGFR, HER3, and HER4, recognize multiple ligands, while HER2 is an orphan receptor. These can from homodimers or heterodimers with HER2 Ligand binding to the ECDs results in receptor dimerization. Through a still unclear mechanism, the ECD dimer communicates with the intracellular domains of the receptor. EGFR family Observation: Different ligands can give different responses in a cell –Biased signaling Epidermal growth factor (EGF) proliferation Transforming growth factor a (TGFa) Epiregulin (EREG) Differentiation Experimental system: Use cells that only have EGFR homodimers GraphicalAbstract Abstract Graphical B Auth Autho Danie Daniel Anato Anatol Diane Diane Corr Corre mark mark.l C C D Highlights Highlights D dd Different EGFR ligands stabilize receptor dimers with distinct Different EGFR ligands stabilize receptor dimers with distinct structures structures dd Epiregulin Epiregulinand andepigen epigeninduce induceweaker weakerand andmore moreshort-lived short-lived EGFR EGFRdimers dimersthan thanEGF EGF dd Weakened Weakeneddimerization dimerizationcauses causessustained sustainedEGFR EGFRsignaling signaling dd Epiregulin Epiregulinand andepigen epigencan caninduce inducecell celldifferentiation differentiation through throughEGFR EGFR InBri Br In Recep Rece princip princ signali signa ds. The ErbB4 ligands BTC, NRG1β, NRG2β, and NRG3 differenstimulate ErbB4 coupling to survival and proliferation in CEM/ formation that exposes a dimerization arm in subdomain II, thereby facilitating dimerization of the extracellular region (Burgess et al., Regulation of the signaling outcomes is due to stability of active receptor complex formation. Called kinetic proof reading. This model assumes a time lag between initial receptor ligand binding and phosphorylation of tyrosines and: Recruitment of downstream signal transducers and full receptor activation. Stable complexes- the receptor becomes fully Transducers activated which leads to activate a negative feedback event. One possibility is recruitment of phosphatase which dephosphorylate and inactivate the receptor Non-stable complexes fall apart prior to activation of negative feedback. The complexes reform keeping the initial signaling event going Ligand stimulation of ErbB receptor tyrosine phosphorylation creates docking sites for numerous signaling effectors. Putative sites of EGFR, ErbB2, ErbB3, and ErbB4 tyrosine horylation are denoted, as well as signaling effectors predicted or shown to bind to these sites of phosphorylation (Rotin et al., 1992; Cohen et al., 1996; Zrihan-Licht et al., 1998; -Licht et al., 1998; Keilhack et al., 1998; Hellyer et al., 2001; Schulze et al., 2005; Kaushansky et al., 2008). The ErbB receptors are not drawn to scale. on of ErbB receptor tyrosine phosphorylation creates docking sites for numerous signaling effectors. Putative sites of EGFR, ErbB2, ErbB3, and ErbB4 tyrosine EGF EGFR HIGH AFFINITY Stable DIMER FORMED Full Act. EGFR Full transducer recruitment Proliferation EGF EGFR HIGH AFFINITY Stable DIMER FORMED Full Act. Full transducer recruitment EGFR Proliferation Phosphatase Activated When more EGF binds EGFR Phosphorylation and signaling inhibited EGF EGFR HIGH AFFINITY Stable DIMER FORMED New EGF Full Act. Full transducer recruitment EGFR Proliferation Phosphatase activated When more EGF binds EGFR Phosphorylation and signaling inhibited EGFR No phosphorylation and signaling Why phosphatase as negative event? SomeEGFR groups Ligands show inhibition of phosphatase shifts Differentially Stabilize Signaling from transient to sustained Dimers to Specify Signaling Kinetics Graphical Abstract Recepto Authors Daniel M. Freed, Nicholas J. Anatoly Kiyatkin,..., Daniel J Diane S. Lidke, Mark A. Lem Correspondence [email protected] In Brief Receptor tyrosine kinases o principles of biased agonism signaling outputs. Highlights d Different EGFR ligands stabilize receptor dimers with distinct structures d Epiregulin and epigen induce weaker and more short-lived EGFR dimers than EGF d Weakened dimerization causes sustained EGFR signaling d Epiregulin and epigen can induce cell differentiation through EGFR ds. The ErbB4 ligands BTC, NRG1β, NRG2β, and NRG3 differenstimulate ErbB4 coupling to survival and proliferation in CEM/ formation that exposes a dimerization arm in subdomain II, thereby facilitating dimerization of the extracellular region (Burgess et al., Regulation of the signaling outcomes is due to stability of active receptor complex formation. Called kinetic proof reading. This model assumes a time lag between initial receptor ligand binding and phosphorylation of tyrosines and: Recruitment of downstream signal transducers and full receptor activation. Stable complexes- the receptor becomes fully Transducers activated which leads to activate a negative feedback event. One possibility is recruitment of phosphatase which dephosphorylate and inactivate the receptor Non-stable complexes fall apart prior to activation of negative feedback. The complexes reform keeping the initial signaling event going Ligand stimulation of ErbB receptor tyrosine phosphorylation creates docking sites for numerous signaling effectors. Putative sites of EGFR, ErbB2, ErbB3, and ErbB4 tyrosine horylation are denoted, as well as signaling effectors predicted or shown to bind to these sites of phosphorylation (Rotin et al., 1992; Cohen et al., 1996; Zrihan-Licht et al., 1998; -Licht et al., 1998; Keilhack et al., 1998; Hellyer et al., 2001; Schulze et al., 2005; Kaushansky et al., 2008). The ErbB receptors are not drawn to scale. on of ErbB receptor tyrosine phosphorylation creates docking sites for numerous signaling effectors. Putative sites of EGFR, ErbB2, ErbB3, and ErbB4 tyrosine Partial transducer recruitment Act. EREG EGFR LOW AFFINITY UNSTABLE DIMER EGFR Differentiation DIMER FALLS APART SIGNALING STOPS Partial transducer recruitment Act. EREG EGFR LOW AFFINITY UNSTABLE DIMER EGFR Differentiation DIMER FALLS APART SIGNALING STOPS No phosphatase activated Partial transducer recruitment Act. EREG EGFR EGFR LOW AFFINITY UNSTABLE DIMER Differentiation DIMER FALLS APART SIGNALING STOPS No phosphatase activated Partial transducer recruitment Act. “New” EREG Activates Signaling again EGFR EGFR Differentiation Conformation model for biased signaling by EGFR Ligands Binding of different ligand results in changes of JM coilcoil dimer Page 32 The ligands EREG,TGFa that induce the TGFa type coil-coil dimer are more likely to induce differentiation Page 32 than EGF that induce nase (RTK); Epidermal Growth Factor Receptor (EGFR); Cancer; EGF-type coil-coil main; Juxtamembrane Domain; Ligand-Induced Dimerization; Signal ery dimer Figure 1. Monitoring growth factor-dependent assembly at the TM-JM junction using bipartite tetracysteine display Conformation model for biased signaling by EGFR Ligands How the differences in the JM coil-coil domain results in differences in signaling has not been determined. May cause changes in kinase domain that alters C-terminal phosphorylation and recruitment of transducers Lecture 5:-Nuclear receptors Super-family of nuclear receptors 2E Table I. Nuclear Receptor Superfamily Family 0B 1A 1B 1C 1D 1F 1H 1I 2A 2B 2C 2E 2F Common name Dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, Gene 1 Short heterodimeric partner Thyroid hormone receptor-α Thyroid hormone receptor-β Retinoic acid receptor-α Retinoic acid receptor-β Retinoic acid receptor-γ Peroxisome proliferator-activated receptor-α Peroxisome proliferator-activated receptor-β Peroxisome proliferator-activated receptor-γ Reverse-Erb-α Reverse-Erb-β Retinoic acid-related orphan-α Retinoic acid-related orphan-β Retinoic acid-related orphan-γ Farnesoid X receptor Farnesoid X receptor-β Liver X receptor-α Liver X receptor-β Vitamin D receptor Pregnane X receptor Constitutive androstane receptor Hepatocyte nuclear Factor-4-α Hepatocyte nuclear Factor-4-γ Retinoid X receptor-α Retinoid X receptor-β Retinoid X receptor-γ Testicular Receptor 2 Testicular Receptor 4 Tailless homolog orphan receptor Photoreceptor-cell-specific nuclear receptor Chicken ovalbumin upstream Abbreviation Gene name DAX1 NR0B1 Orphan SHP TRα TRβ RARα RARβ RARγ PPARα PPARβ PPARγ REV-ERBα REV-ERBβ RORα RORβ RORγ FXRα FXRβ LXRα LXRβ VDR PXR NR0B2 THRA THRB RARA RARB RARG PPARA PPARD PPARG NR1D1 NR1D2 RORA RORB RORC NR1H4 NR1H5P NR1H3 NR1H2 VDR NR1I2 NR1I3 HNF4A HNF4G RXRA RXRB RXRG NR2C1 NR2C2 NR2E1 NR2E3 NR2F1 Orphan Thyroid hormones Thyroid hormones Retinoic acids Retinoic acids Retinoic acids Fatty acids Fatty acids Fatty acids Heme Heme Sterols Sterols Sterols Bile Acids Orphan Oxysterols Oxysterols 1α,25-dihydroxyvitamin D3 Endobiotics and xenobiotics Xenobiotics Fatty acids Fatty acids 9-Cis retinoic acid 9-Cis retinoic acid 9-Cis retinoic acid Orphan Orphan Orphan Orphan Orphan HNF4α HNF4γ RXRα RXRβ RXRγ TR2 TR4 TLX PNR COUP-TFα Ligand 2F 3A 3B 3C 4A 5A 6A Testicular Receptor 4 Tailless homolog orphan receptor Photoreceptor-cell-specific nuclear receptor Chicken ovalbumin upstream promoter-transcription factor α Chicken ovalbumin upstream promoter-transcription factor β Chicken ovalbumin upstream promoter-transcription factor γ Estrogen receptor-α Estrogen receptor-β Estrogen-related receptor-α Estrogen-related receptor-β Estrogen-related receptor-γ Androgen receptor Glucocorticoid receptor Mineralocorticoid receptor Progesterone receptor Nerve growth Factor 1B Nurr-related Factor 1 Neuron-derived orphan Receptor 1 Steroidogenic Factor 1 Liver receptor Homolog-1 Germ cell nuclear factor TR4 TLX PNR COUP-TFα NR2C2 NR2E1 NR2E3 NR2F1 Orphan Orphan Orphan Orphan COUP-TFβ NR2F2 Orphan COUP-TFγ NR2F6 Orphan ERα ERβ ERRα ERRβ ERRγ AR GR MR ESR1 ESR2 ESRRA ESRRB ESRRG AR NR3C1 NR3C2 PR NGF1-B NURR1 NOR-1 SF-1 LRH-1 GCNF PGR NR4A1 NR4A2 NR4A3 NR5A1 NR5A2 NR6A1 Estrogens Estrogens Orphan Orphan Orphan Androgens Glucocorticoids Mineralocorticoids and glucocorticoids Progesterone Orphan Unsaturated fatty acids Orphan Phospholipids Phospholipids Orphan Modular Structure of Nuclear Receptors Like most TFs, nuclear receptors have a modular structure. They all contain highly conserved DNA-binding domains, consisting of C4 zinc finger motifs, located near the middle of the polypeptide chain.