Signal Transduction Pathway.ppt

Signal Transduction Prof. Dr. Deniz KIRAÇ 1 2 Signaling molecule Receptor of target cell Intracellular molecule Signal transduction Biological effect 3 4 Signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another. It is a process of c...

Signal Transduction Prof. Dr. Deniz KIRAÇ 1 2 Signaling molecule Receptor of target cell Intracellular molecule Signal transduction Biological effect 3 4 Signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another. It is a process of converting extracellular signals to a language that inside of the cell can deal with. Most processes of signal transduction involve ordered sequences of biochemical reactions inside the cell, which are carried out by enzymes and activated by second messengers, resulting in a signal transduction pathway. 6 Signaling molecules Signaling molecules, which are released by signalproducing cells, reach and transfer biological signals to their target cells to initiate specific cellular responses. There are two types of signaling molecules. • Extracellular molecules • Intracellular molecules 7 1. Extracellular molecules Steroid hormones Nitric oxide (NO) Neurotransmitters Peptide hormones (insulin, glucagon) Growth factors (EGF, PDGF, cytokines) 8 a) Paracrine signaling • • • • Secreted by common cells. Reach neighboring target cells by passive diffusion. Time of action is short. Such as nitric oxide 9 • Nitric oxide (NO) is a major paracrine signalling molecule in the nervous, immune and circulatory systems. • NO is synthesized from the amino acid arginine by the enzyme nitric oxide synthase. • The major target of NO is guanyl cyclase • NO binds to guanyl cyclase and stimulates the synthesis of cGMP (will be discussed later). • This process induced muscle cell relaxation and blood vessel dilation. 10 b) Endocrine signalling • • • • Secreted by endocrine cells. Reach target cells by blood circulation. Time of action is long. Such as insulin, adrenaline c) Autocrine signalling • Act back to their own cells. • Such as GF, cytokine, interferon, interleukin. 11 12 2. Intracellular molecules • Ca2+ • IP3 • cAMP cGMP • Ras, JAK, Raf 13 Functions of cell surface receptors G-protein-coupled receptors • The largest family of cell surface receptors transmits signal to intracellular targets via the intermeairy action of guanine nucleotide-binding proteins called G proteins. • More than a thousand such G protein-coupled receptors have been identified, including the receptors for eicosanoids, many neurotransmitters, neuropeptides and peptide hormones. 14 G proteins consist of three subunits α,β and γ. • α subunit (38-46 kDa) (at least 20 different genes) • β subunit (37 kDa) (5 different genes) • γ subunit (8 kDa) (12 different genes) 15 Binding of hormone promotes the interaction of the receptor with a G protein. The activated G protein α subunit then dissociates from the receptor and stimulates adenylyl cyclase, which catalyzes the conversion of ATP to cAMP. 16 Regulation of G proteins 17 Some G proteins can also directly regulate ion channels 18 19 • • • • • Neurotransmitters ; Secreted by neuronal cells. Reach another neuron by synaptic gap. Time of action is short. Such as Acetylcholine, dopamine, adrenaline. Neurotransmitters also acts as hormones. 20 21 Receptor protein-tyrosine kinases • In contrast to G protein-coupled receptors, other cell surface receptors are directly linked to intracellular enzymes. • The largest family of these receptors are the receptor proteintyrosine kinases, which phosphorylate their substrate proteins on tyrosine residues. • Eg. Growth factors such as PDGF, EGF 22 Organization of receptor protein-tyrosine kinases 23 Dimerization and autophosphorylation of receptor protein-tyrosine kinases 24 Secondary Messengers • They are special intracellular molecules between the receptor region and the target intracellular affect region. • They cause intracellular chemical changes. 25 • The primary messenger is the extracellular hormone. • Secondary messengers - Ca2+, IP3, cAMP,cGMP– they are secreted when te hormone binds to the extracellular receptor. • Secondary messenger activates or inhibits the other messengers in cytoplasm or nucleus. 26 27 28 29 Nitric Oxide (NO) • NO is a secondary messenger that can behave as a reactive or free radical. • NO can cause the relaxation of vascular smooth muscle cells. • NO can also stimulate macrophages and kills tumor cells and bacteria. • NO binds to the heme region of guanyl cyclase and induces the Guanylyl cyclase activity 10 times. 30 Ca2+ • Both G-protein and tyrosine kinase pathways can utilize calcium as a second messenger • Calcium concentrations in cytoplasm are normally very low • Calcium come from the extracellular environment and from the ER • Calcium is exported by calcium pumps within cell, to keep internal calcium levels low 31 32 cAMP (cyclic AMP) Adenylate Cyclase (Adenylyl Cyclase) catalyzes: ATP cAMP + PPi cAMP N N Binding of certain hormones (e.g., epinephrine) to the outer surface of a cell activates Adenylate Cyclase to form cAMP within the cell. Cyclic AMP is thus considered to be a second messenger. NH2 N N H2 5' C 4' O O O H H 3' P O H 1' 2' H OH O- 33 Effects of cAMP In skeletal muscle and liverglycogenolysis Cardiac muscle- strengthens muscle contraction Smooth muscle- inhibits contraction Intestinal epithelium- movement of salt and water into gut 34 One response to cAMP-mediated signaling 35 O P ro te in K in a s e O H + ATP P ro te in P ro te in O P O + ADP O Pi H 2O P ro te in P h o s p h a ta s e A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. A protein phosphatase catalyzes removal of the Pi by hydrolysis. (Proteins are activated by kinase activity and inactivated by protein phosphatases) 36 O P ro te in K in a s e O H + ATP P ro te in P ro te in O P O + ADP O Pi H 2O P ro te in P h o s p h a ta s e Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example: Some protein kinases are activated by Ca++-calmodulin. Protein Kinase A (cAMP dependent kinase) is activated by cAMP. 37 cAMP NH2 Phosphodiesterase enzymes catalyze: N N cAMP + H2O AMP The phosphodiesterase that cleaves cAMP is activated by phosphorylation catalyzed by Protein Kinase A. N N H2 5' C 4' O O O H H 3' P O H 1' 2' H OH O- 38 cGMP (cyclic GMP) • cGMP is also an important second messenger. • It is formed from GTP by guanylyl cyclase and degraded to GMP by a phosphodiesterase. • Guanylyl cyclases are activated by nitric oxide, carbon monoxide as well as peptid ligands. • Stimulation of guanylyl cyclases leads to elavated level of cGMP, which then mediate biological responses, such as blood vessel dilation. • The best characterized role of cGMP is in the vertabrate eye, where it serves as the second messenger responsible for converting the visual signals received at light to nerve impulses. 39 • Absorption of light by retinal activates the G-protein-coupled receptor rhodopsin. • When rhodopsin is activated, it activates G protein transducin. • Α subunit of transducin stimulates the activity of cGMP phosphodiesterase, leading to a decrease in the intracellular level of cGMP. • This change in cGMP level in retinal rod cells is translated to a nerve impulse by a direct effect of cGMP on ion channels in the plasma membrane. 40 G Protein Signal Cascade Most signal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. Only signal molecules able to cross the plasma membrane (e.g., steroid hormones) interact with intracellular receptors. A large family of cell surface receptors have a common structural motif, 7 transmembrane ahelices. Rhodopsin was the first of these to have its 7helix structure confirmed by X-ray crystallography. Rhodopsin PDB 1F88 41 G-proteins are heterotrimeric, with 3 subunits α,β,γ A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gsα. Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon. 42 hormone signal outside GPCR The α subunit of a Gprotein (Gα) binds GTP, & can hydrolyze it to GDP + Pi. plasma membrane  AC                       GTP GDP GTP GDP cytosol ATP cAMP + PP i α & γ subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site. 43 hormone signal outside GPCR plasma membrane  AC                         GDP GTP GTP GDP cytosol ATP cAMP + PP i The sequence of events by which a hormone activates cAMP signaling: 1. Initially Ga has bound GDP, and α,β,γ subunits are complexed together. Gβ,γ the complex of β and γ subunits, inhibits Ga. 44 hormone signal outside GPCR plasma membrane  AC GDP GTP GTP GDP cytosol ATP cAMP + PP i 2. Hormone binding, usually to an extracellular domain of a 7-helix receptor (GPCR), causes a conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the membrane. The nucleotide-binding site on Ga becomes more accessible to the cytosol, where [GTP] > [GDP]. Ga releases GDP & binds GTP (GDP-GTP exchange). 45 hormone signal outside GPCR plasma membrane  AC GDP GTP GTP GDP cytosol ATP cAMP + PP i 3. Substitution of GTP for GDP causes another conformational change in Ga. Ga-GTP dissociates from the inhibitory βγ complex & can now bind to and activate Adenylate Cyclase. 46 hormone signal outside GPCR plasma membrane  AC GDP GTP GTP GDP cytosol ATP cAMP + PP i 4. Adenylate Cyclase, activated by the stimulatory synthesis of cAMP. Ga-GTP, catalyzes 5. Protein Kinase A (cAMP Dependent Protein Kinase) catalyzes transfer of phosphate from ATP to serine or threonine residues of various cellular proteins, altering their activity. 47 TURN OFF the signal: 1. Ga hydrolyzes GTP to GDP + Pi. (GTPase). The presence of GDP on Ga causes it to rebind to the inhibitory βγ complex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterases catalyze hydrolysis of cAMP AMP. 48 Gene expression in response to a ligand 49 • In many animal cells, increases in cAMP activate the transcription of spefic target genes that contain a regulatory sequence called the cAMP response element (CRE). • In this case, the signal is carried from the cytoplasm to the nucleus by protein kinase A. • Within the nucleus, protein kinase A phosphorylates a transcription factor called CREB (for CRE-binding protein), leading to the activation of cAMP-inducible genes. • Such regulation of gene expression by cAMP plays important roles in controlling the proliferation, survival and differentiation of a wide variety of animal cells. 50 51 Phosphatidylinositol Signal Cascades O O R1 C H2 C O O C CH H2 C R2 O O P O O OH 2 phosphatidylinositol H H 1 6 H OH OH H 3 H 4 OH 5 H OH Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol. 52 O O R1 C H2C O O C CH H2C R2 O O P O O OH 2 H PIP 2 phosphatidylinositol4,5-bisphosphate H 1 H OH 3 H 6 OH H 4 OPO 3 2 5 H OPO 3 2 Kinases sequentially catalyze transfer of P i from ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is a minor component of the plasma membrane, localized to the inner part of the phospholipid bilayer. PIP2 is cleaved by the enzyme Phospholipase C. 53 O PO 3 2  H OH 2 H 1 6 H OH OH H 3 H O PO 3 2  5 H 4 O PO O R1 C H2C O O C R2 CH 2 IP 3 ino sitol-1 ,4 ,5 -trisph o sp hate 3 O H2C OH diacylglycerol Cleavage of PIP2, catalyzed by Phospholipase C (various hormones and growth factors stimulate the hydrolysis of PIP2 by phospholipase C), yields 2 second messengers:  inositol-1,4,5-trisphosphate (IP3)  diacylglycerol (DAG). 54 DAG and IP3 stimulate signalling pathways (protein kinase C and Ca2+ mobilization, respectively) Hydrolysis of PIP2 55 IP3 acts to release Ca2+ from endoplasmic reticulum by binding to receptors that are ligand-gated Ca2+ channels. 56 Ca++ Ca++-release channel IP3 Ca ATP calmodulin Ca ++ endoplasmic reticulum Ca++-ATPase ++ ADP + Pi Ca2+ stored in the ER is released to the cytosol, where it may bind calmodulin (Ca2+ binding protein), or help activate Protein Kinase C. Signal turn-off includes removal of Ca2+ from the cytosol via Ca2+-ATPase pumps, & degradation of IP3. 57 Ras, Raf and MAP kinase pathway • The MAP kinase pathway refers to a cascade of protein kinases that are highly conserves in evolution and play central roles in signal transduction. • The central elements in the pathway are a family of protein-serine/threonine kinases called the MAP kinases (mitogenactivated protein kinases) that are activated in response to a variety of growth factors and other signalling molecules. • In higher eukaryotes, MAP kinases are ubiquitous regulators of cell growth and differentiation. 58 • The MAP kinases that were initally charactarized in mammalian cells belong to the ERK (extracellular signal-regulated kinase) family. • ERK activation plays a central role in signalling cell proliferation induced by growth factors that act through either protein-tyrosine kinase or G protein-coupled receptors. • Activation of ERK is mediated by Ras and Raf. Activation of these kinases activates a second protein kinase called MEK (MAP kinase/ERK kinase). • MEK activates ERK family by phosphorylation. • ERK then phosphorylates a variety of nuclear and cytoplasmic target proteins. 59 Activation of the ERK MAP Kinases 60 Activated ERK translocates to the nucleus, where it phosphorylates the transcription factor Elk-1. Elk-1 binds to the serum response element (SRE) in a complex with serum response factor (SRF). Phosphorylation stimulates the activity of Elk-1 as a transcriptional activator, leading to immediate-early gene induction. 61 Ras proteins are guanine nucleotide binding proteins that function analogously to the α subunits of G proteins, alternating between inactive GDP-bound and active GTPbound forms. Regulation of RAS proteins 62 JAK/STAT Pathway • JAK/STAT Pathway provides a much more immediate connection between protein-tyrosine kinases and transcription factors than MAP kinase pathway. • In this pathway protein-tyrosine phosphorylation directly affects transcription factor localization and function. • The key elements in this pathway are the STAT proteins (signal transducers and activators of transcription). • In unstimulated cells, STAT proteins are inactive in the cytosol. 63 • Stimulation of cytokine receptors leads to the binding of STAT proteins, where they are phosphorylated by the receptorassociated JAK protein tyrosine kinases. • The phosphorylated STAT proteins then dimerize and translocate to the nucleus, where they activate the transcription of target genes. The JAK/STAT Pathway 64