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Signal Transduction 2 Molecular Biology of Cancer 1 Signal Transduction by the Mitogenic Pathways Cells in an organism receive a variety of extracellular stimuli for cell proliferation. Signal transduction is the intracellular event that convey extracellular stimuli into specific cellular responses protein-protein interaction Phosphorylation Molecular Biology of Cancer 2 The MAPK signalling pathways The best characterized mitogenic pathway is: the mitogen-activated protein kinase (MAPK) cascades also called extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) Many growth stimulation converges on the kinase cascade that activates the MAPK Molecular Biology of Cancer 3 The RAS-activated MAPK pathway The first example where all the steps in a complete signalling cascade from the cell surface receptor PTK, to the nuclear transcription is known RAS RAF MEK MAPK Molecular Biology of Cancer 4 Ras (from rat sarcoma) is a GEF guanine nucleotide exchange factor GTPase activity Molecular Biology of Cancer 5 Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2 (a SH2- and SH3-containing protein): binds to the receptor phosphotyrosine via its SH2 domain constitutively binds via its SH3 to the prolinerich sequence in the C-terminus of SOS (a guanine nucleotide exchange factor) SOS is recruited to the close proximity of RAS in the membrane P RAS P 14-3-3 SOS P GTP GDP RAF P P GRB2 RAS becomes activated by exchanging GDP for GTP The active RAS-GTP: interacts with the N-terminal regulatory region of the RAF (serine/threonine protein kinase) RAF recruited to the membrane and changes its conformation phosphorylation of RAF and binding to the scaffold protein 14-3-3 Molecular Biology of Cancer 6 Molecular Biology of Cancer 7 Activated RAF: activates MEK (also called MAPK kinase; a dual specificity kinase) by phosphorylation on two conserved serine residues in MEK. Activated MEK: activates MAPK (a serine/threonine protein kinase) by phosphorylation of conserved threonine and tyrosine residues. P RAS P 14-3-3 SOS P GRB2 it is also translocated into the nucleus (within minutes) where it phosphorylates nuclear transcription factors. Transcription of genes important for cell proliferation. Substrates RAF P P Activated MAPK: phosphorylates a number of substrates in the plasma membrane and the cytoplasm; GTP GDP P P P MEK P P MAPK Substrates Substrates P P Molecular Biology of Cancer P P MAPK 8 Molecular Biology of Cancer 9 Substrates of MAPK MAPK phosphorylates: In cytoplasm: MAPK phosphorylates its upstream components in a negative feedback loop MAPK phosphorylates SOS, RAF, MEK inhibition of MAP kinase pathway. In nucleus: MAPK phosphorylates a number of transcription factors (e.g. Elk1) increase transcription (e.g. of c-Fos mRNA). Many other substrates: of MAPK probably unknown identification is difficult as in the case of CDKs Molecular Biology of Cancer 10 The MAPK signalling pathways It should be noted that the RASRAFMEKMAPK pathway is only one example of so called “MAPK pathways” Two other mammalian MAPK pathways involving JNK1 and p38, are involved in stress responses (they are also “MAPK pathways). JNK pathway: a family of MAPK relatives known as JNKs (also called stressactivated protein kinase (SAPKs) become activated in response to extracellular stresses like cycloheximide treatment, UV irradiation, heat shock, or TNF-a treatment,. Molecular Biology of Cancer 11 RAC1 and CDC42 are two members of the RHO family of GTP-binding proteins. RAC1 and CDC42 are mainly activated by stress response independent of RAS RAC1 can also be activated by RAS (minor pathway) explaining why receptor PTK can sometimes contribute to JNK activation. STRESS GTP RAC1/CDC42 PAK P P MEKK1-3 GTP-bound form of RAC1 and CDC42 bind and activate the serine/threonine P P protein kinase PAK, PKN, and PtdIns MEK4 kinases. these kinases phosphorylate and PAK: p21-activated protein kinase activate MEKK1-3 PKN: protein kinase N MEKK1-3 phosphorylate and activate PtdIns kinase: phosphatidylinositol kinase MEK4 (also called JNKK) (~ MEK in MAPK pathway; 45% identical in sequence with MEK) Molecular Biology of Cancer 12 MEK4 phosphorylates JNK at two similar sites as in ERK (but T-P-Y in JNK instead of T-E-Y in MAPK) STRESS i.e. conservation between the ERK and JNK pathways at the level of proteins and mode of regulation! JNK translocation into the nucleus phosphorylation of the transcription factor c-JUN at the Nterminal residues (Ser63 and Ser73) activation of transcription by cJUN GTP RAC1/CDC42 PAK P P MEKK1-3 P P MEK4 P P JNK P P JNK P c-JUN Molecular Biology of Cancer 13 STRESS P RAS P SOS P GTP 14-3-3 RAF P GTP RAC1/CDC42 P P MEKK1-3 PAK GRB2 P P MEK P P MAPK P P MEK4 P P JNK Molecular Biology of Cancer 14 Generic pathway ERK/MAP kinase pathway JNK/SAPK pathway Proliferation/differentiation p38 pathway Stress responses Receptor PTK GRB2/SOS RAS RAC/CDC42 PAK MAPKKK RAF1 MEKK1-3 TAK MAPKK MEK1,2 MEK4 MEK3,6 MAPK ERK1,2 JNK/SAPK p38 Molecular Biology of Cancer 15 Specificity of MAP kinase pathways It seems JNK and ERK pathways are biologically distinct. However, they are both protein kinases with similar substrate specificity most in vitro substrates are the same for both. Yet these pathways must result in unique transcriptional activity - because stress and mitogen must elicit different responses There are at least five parallel MAP kinase pathways in mammalian cells. How is specificity achieved? Molecular Biology of Cancer 16 Specificity of MAP kinase pathways One way is by scaffold proteins. Molecular Biology of Cancer 17 G Protein-Linked Receptors G protein-linked receptors compose the largest family of cell-surface receptors: >100 members in mammals include: light-activated receptors (rhodopsins) in the eye odorant receptors in the nose receptors for various hormones and neurotransmitters Molecular Biology of Cancer 18 G Protein-Linked Receptors A number of different hormones mediate biological responses by binding to G protein-linked receptors - and a-adrenergic receptors Muscarinic cholinergic receptors Vasopressin (ADH) Angiotensin II Serotonin Substance P Dopamine Lutenizing hormone (LH) Follicle-stimulating hormone (FSH) Thyroid stimulating hormone (TSH) Platelet-activating factor Prostaglandins Rhodopsin Molecular Biology of Cancer 19 G Protein G proteins are guanine nucleotide-binding proteins composed of a-, -, and -subunits The a-subunit is unique to each type of G protein, but all the and -subunits for all the different types of G proteins are very similar The a-subunit binds to the guanine nucleotide (GDP or GTP) So far we have identified a Gs (as), a Gi (ai), a Gq (aq) and a Gt (at) protein Distinct from the monomeric GTPbinding proteins GTPase e.g. Ras Molecular Biology of Cancer 20 Molecular Biology of Cancer 21 G Protein-Linked Receptors Seven-spanning G protein-linked receptors: contain seven stretches of ~22-24 hydrophobic residues, forming seven transmembrane a helices G protein binds to: 1.the loop between a helices 5 and 6; and 2.the C-terminal region Molecular Biology of Cancer 22 G protein acts as an on/off switch No ligand G protein binds GDP inactive Ligand binding to receptor G protein binds GTP active Activated G protein binds to and activates an effector enzyme, which catalyzes the formation of a secondary messenger. Hydrolysis of GTP to GDP converts G protein back to inactive state. Molecular Biology of Cancer 23 Example of G protein-linked receptor Adrenaline receptor 1. Hormone binding to a- and -adrenergic receptors. 2. The receptor interacts with G protein 3. Activation/inhibition of adenylate cyclase (effector enzyme). 4. Increase/decrease in intracellular cAMP (secondary messenger). Molecular Biology of Cancer 24 Binding of hormone to -adrenergic receptors conformational change in loop between helices 5 and 6 bind to Gs in such a way that GDP is displaced and GTP is bound G and G are dissociated from Gsa-GTP Gsa-GTP is able to bind to and activate adenylate cyclase activated adenylate cyclase can then produce cAMP from ATP GTP bound to Gsa is quickly hydrolysed to GDP (seconds) association of G and G with Gsa-GDP inactivation of adenylate cyclase G Gb GTP GSa GTP GDP G Gb AC GSa GSa GDP GTP GDP Molecular Biology of Cancer cAMP + PPi ATP 25 Amplification of signal 1. Activated Gsa-GTP can diffuse rapidly one activated receptor can activate many Gs. 2. One Gsa-GTP can bind to only one adenylate cyclase but this can catalyze the synthesis of many cAMP. Molecular Biology of Cancer 26 Some bacterial toxins irreversibly modify G proteins Cholera toxin: a peptide produced by the bacterium Vibrio cholerae, causes serious diarrhea death by dehydration. Irreversibly modifies Gsa (at Arg174, which is located near the GTP-binding site in Gsa) modified Gsa can bind GTP but cannot hydrolyze it to GDP permanent activation of adenylate cyclase sustained high cAMP level; in intestinal epithelial cells this sustained increase in cAMP causes membrane proteins to allow water efflux into the intestine. Molecular Biology of Cancer 27 Gi may inhibit adenylate cyclase by two mechanisms: 1. The ai-GTP complex interacts with adenylyl cyclase, inhibiting its activity 2. Adenylyl cyclase activity is further reduced by increasing the amount of -subunits; this allows them to interact with as-subunits preventing activation of adenylyl cyclase Molecular Biology of Cancer 28 cAMP as a Second Messenger The main target of cAMP in the cell is cAMP-dependent kinase (PKA). PKA is a serine/threonine protein kinase. Inactive conformation: a dimer of PKA binding to two regulatory subunits. Each regulatory subunit contains two cAMP binding sites When cAMP binds cooperatively to the regulatory subunits regulatory subunits dissociate from the PKA PKA becomes activated. Molecular Biology of Cancer 29 PKA substrates PKA catalyzes phosphorylation and activation of hormone-sensitive lipase, cholesteryl esterase, & glycogen phosphorylase, and inhibits glycogen synthase cAMP also (through PKA) regulates gene transcription Phosphoenolpyruvate carboxykinase Tyrosine aminotransferase Human glycoprotein hormone a-subunit gene Preprosomatostatin Vasoactive intestinal polypeptide A surfactant protein, SP-A Several isoforms of cytochrome P450 Molecular Biology of Cancer 30 Examples of PKA substrates CRE-binding protein (CREB) - a transcription factor (for DNA sequence called cAMP response elements) phosphorylation of CREB by PKA stimulates its transcription activity. Molecular Biology of Cancer 31 The Gq protein-linked receptors and Ca2+ Ca2+ is an important intracellular second messenger. [Ca2+] in the cytosol is low (10-7 M) [Ca2+] outside the cell is high (10-3 M) [Ca2+] in ER also high. Extracellular signals open Ca2+ channels in plasma / ER membranes Ca2+ rushes into the cytosol increase Ca2+ Ca2+dependent responses. Molecular Biology of Cancer 32 Control of cytosolic calcium Ca2+-ATPase in plasma membrane and ER membrane pumps Ca2+ out of the cytosol (use ATP as energy) into the extracellular space and the ER respectively. Normally, free [Ca2+] changed from ~10-7 M in resting cells to ~5x10-6 M in stimulated cells. If Ca2+ pumps are defective and the free [Ca2+] in the cytosol gets to >10-5 M, a low affinity, high capacity Ca2+ pump in the inner mitochondrial membranes kicks in and pump Ca2+ into the mitochondria (uses electrochemical gradient as energy). Molecular Biology of Cancer 33 Molecular Biology of Cancer 34 Adapted from Molecular Biology of the Cell Overview G G DAG: Diacylglycerol PLC- Gqa GDP Activates PKC IP3 :Inositol triphosphate Release Ca2+ from ER 1. Extracellular signaling molecules binds to G protein-linked receptor in the plasma membrane. Activation of a G protein Gq 2. Activation of phospholipase C- 3. Cleaves phosphatidylinositol bisphosphate (PIP2) into two products: 2 different signal transduction pathways Molecular Biology of Cancer 35 Phosphatidylinositol (PI) is a minor phospholipid in cell membranes; PIP2 is a phosphorylated derivative of PI - located in the inner half of the plasma membrane lipid bilayer. Molecular Biology of Cancer 36 Molecular Biology of Cancer 37 IP3 activates Ca2+ release from the ER IP3 binds to the IP3-gated Ca2+ release channels in the ER membrane release Ca2+ into the cytosol (by gradient). Depleted Ca2+ store promotes influx of extracellular Ca2+ via membrane channels (signals by the release Ca2+ or factor from empty store?) Molecular Biology of Cancer 38 DAG IP3 Enzymes (e.g. myosin light-chain kinase, phosphorylase kinase, Ca2+-calmodulin kinase II etc) Membrane transport proteins (e.g. Ca2+-ATPase on plasma membrane Calmodulin ER Ca2+ 4 high-affinity Ca2+-binding sites IP3-gated Ca2+ release channels Molecular Biology of Cancer 39 Calmodulin Calmodulin is a polypeptide that undergoes a conformational change when it binds to calcium The conformational change allows the calmodulin effect on cellular proteins Many effects of Ca2+ are mediated by Ca2+/calmodulin-dependent kinases (CaMkinases). The best studied example of CaM-kinase is CaM-kinase II. CaM-kinase II is found in all animal cells but is especially enriched in the nervous system. Molecular Biology of Cancer 40 Molecular Biology of Cancer 41 CaM kinases Functions of CaM-kinase II: Molecular memory device switching to active state when exposed to Ca2+/calmodulin. remains active by autophosphorylation (i.e. remains active even when Ca2+ is removed) inactivated only when the phosphatase overwhelms the autophosphorylation) important in memory (mice lacking CaM-kinase II have defects in remembering where things are in space) Molecular Biology of Cancer 42 Molecular Biology of Cancer 43 Termination of Ca2+ response 1. Breakdown of DAG 2. Further phosphorylation of PIP2 3. IP3 is dephosphorylated and inactivated by phosphatases. (sometimes it is further phosphorylated to IP4 to mediate other responses) 4. Ca2+ is pumped out of the cell by Ca2+-ATPase 5. Phosphatases which inactivate CaM-kinase II Molecular Biology of Cancer 44 Diacylglycerol (DAG) DAG is also produced when PLC is activated has two signaling roles: 1. Cleave further to release arachidonic acid (as a messenger or for the synthesis of eicosanoids); 2. along with Ca2+ activates PKC (a seine/threonine kinase) DAG increases the affinity of PKC for Ca2+ and for phospholipids Phospholipid and Ca2+ binding activate PKC which phosphorylates serine and threonine residues of certain cellular proteins Molecular Biology of Cancer 45 Ca2+ induces PKC to move from cytosol to plasma membrane PKC is activated by Ca2+, DG (and a membrane phospholipid phosphatidylserine) at the plasma membrane Activated PKC then phosphorylates several substrates DG IP3 P A P B Molecular Biology of Cancer P C PKC 46 Examples of PKC substrates 1. Ion channels in nerve cells changes their activity changes the excitability of nerve cells the highest concentration of PKC is found in the brain 2. PKC phosphorylates and activates protein kinase cascades (e.g. MAPK cascade) transcription of genes (those regulated by JUN, FOS etc.) PKC activates AP-1, a transcription factor made up of one c-Fos and one cJun (each of which is a proto-oncogene) AP-1 recognizes and binds to a DNA sequence similar to CREB PKC is thought to activate AP-1 by activating a phosphatase that dephosphorylates one part of AP-1 and a kinase that phosphorylates a different part of AP-1 3. PKC phosphorylates I-B release NF-B NF-B travel to the nucleus and activate transcription Molecular Biology of Cancer 47 DG PKC P P MAPK IkB Gene 1 NFkB P Activates transcription Nucleus Gene 2 Molecular Biology of Cancer 48 Receptor crosstalk Molecular Biology of Cancer 49 Receptor-linked Tyr kinases This is a common motif. It is called the Jak/STAT pathway for gene regulation. Molecular Biology of Cancer 50 Signal transduction by nuclear receptors Molecular Biology of Cancer 51 Steroid Hormone Receptors The consensus sequence of DNA binding sites of glucocorticoid-receptors (called response elements) = 6 bp inverted repeats separated by any 3 bp. This suggests that these steroid receptors bind to DNA as symmetrical dimers (later confirmed by X-ray crystallography). e.g. Glucocorticoid receptor response element: 5’-AGAACA(N)3TGTTCT-3’ 3’-TCTTGT(N)3ACAAGA-5’ Molecular Biology of Cancer 52 Glucocorticoid receptor - a C4 zinc-finger homodimer Molecular Biology of Cancer 53 Steroid Hormone Receptors Different hormone receptors are conserved in their amino acid sequences and functional domains - all contain: 1. An unique N-terminal region that contains the activation region 2. DNA binding domain 3. Hormone binding domain 1 2 N 3 C Molecular Biology of Cancer 54 GLU EST Hormone BD Hormone BD AD AD DNA BD DNA BD Molecular Biology of Cancer 55 If the DNA binding domain of glucocorticoid receptor is replaced with the similar region of the estogen receptor, the recombinant protein binds to estogen response elements in DNA in response to glucocorticoid. GLU Hormone BD AD DNA BD Molecular Biology of Cancer 56 Regulation of steroid receptors by hormones The hormone binding domain inhibits transcription activation in the absence of hormone. Evidence: - deletion of hormone binding domain of glucocorticoid receptor constitutive activity (even in the absence of hormone). Hormone BD AD DNA BD AD DNA BD Inactive w/o hormone Release inhibition Molecular Biology of Cancer 57 Model Absence of hormone: the receptor is anchored in the cytoplasm by binding to inhibitor proteins no binding to response element no transcription activation Binding to hormone: the receptor is released from the inhibitor protein hormone-receptor complex enter nucleus binds response element and transcription activation Molecular Biology of Cancer 58 The proteins that retain hormone receptor in the cytoplasm are likely to be proteins known as molecular chaperones - which includes heat-shock protein (HSP90) HSP90 masked the nuclear localization signal (NLS) in the absence of hormone Hsp90 NLS Hormone BD AD DNA BD GLU Nuclear membrane Molecular Biology of Cancer 59