1. Signal transduction pathways II: Enzyme-linked receptors Nuclear receptors Stochasticity in signalling.

2. Enzyme-linked receptors signalling through growth factors, hormones, cytokines acting locally at very low concentrations (10 -9 – 10 -11 M) response is usually slow (min-hours) and requires many intracellular steps, leading to changes in gene expression 1. receptor tyrosin kinases (RTKs) 2. tyrosin kinases associated receptors 3. receptor serin/threonin kinases 4. histidin kinase associated receptors 5. receptor guanylyl cyclases

3. 1. Receptor tyrosin kinases (RTKs) Ligands - EGF, PDGF, HGF, NGF, insulin, ephrins... Signalling through monomeric GTPases or through lipid modifications by PI3K Figure 15-52 Molecular Biology of the Cell (© Garland Science 2008)

4. Table 15-4 Molecular Biology of the Cell (© Garland Science 2008)

5. Figure 15-16c Molecular Biology of the Cell (© Garland Science 2008) - ligand is a dimer (PDGF) - ligand is monomer but binds to TM heparan sulphate proteoglycans to form multimers (FGF) - clustering of TM ligand in a neighbouring cell (ephrins) - receptor is a multimer to start with (insulin) - ligand is monomer but binds two receptors simultaneously (EGF) 1. Clusterring of receptors

6. Figure 15-53a Molecular Biology of the Cell (© Garland Science 2008) 2. induced proximity trans/auto phosphorylation

7. Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008) kinase-dead dimerization OK ? How would you create a DN form of enzyme associated receptor?

8. 3. transient assembly of intracellular signalling complexes docking via SH2 domains, PTB domains, PH domains and others some are kinases (Src, PI3K) some are adaptor proteins (Grb) that connect to a protein with enzymatic a. some are inhibitors (e.g. c-Cbl, monoUbiq. and endocytosis)

9. SH2 domain, PTB domain – recognize phosporylated tyrosines SH3 domain – recognizes proline rich sequences PH domain – recognizes modified lipids SH2 domain

10. 4. spreading of the intracellular signalling events Ras family of monomeric GTPases Only Ras and Rho signal through cell surface receptors Often tethered to membrane via lipid anchor

11. Figure 15-19 Molecular Biology of the Cell (© Garland Science 2008) Ras: hyperactive forms such as Ras V12 dominant, constitutively active resistant to inactivation by GAP (no GTP hydrolysis) Ras required for cell proliferation; mutant in 30% of human cancers RTKs activate a Ras-GEF or inhibit a Ras-GAP GEF – guanine exchange factor GAP – GTPase activating proteins

12. Figure 15-56 Molecular Biology of the Cell (© Garland Science 2008) EGFR signalling: lesson from the Drosophila eye 760 ommatidia: 8 photoreceptor cells (R1-8) and 11 support cells, pigment cells, and a cornea

13. Dominant allele of EGFR, constitutively active

14. EGFR required: early for disc proliferation spacing of R8 survival differentiating cells recruitment of all non-R8... but not for: R8 differentiation furrow progression Same for Ras

15. Figure 15-57 Molecular Biology of the Cell (© Garland Science 2008) Grb2 in mammals

16. Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008) mitogen activated kinase (extracellular signal regulated kinase) ? What is the advantage to have many components on the way?

17. EGFR in mammals – ERBB1(HER1) http://www.nature.com/scitable/topicpage/activation-of-erbb-receptors-14457210

18. ERBB family of tyrosin kinases 4 receptors ERBB1-4 (HER1-4) different numbers of phosphorylation sites on their C-termini cellular outcome depends on the dimerization partners and ligands exists as homo or heterodimers Ligands : growth factors (EGF family, NGF,TGF-a)

20. Rho family of monomeric GTPases - regulate actin and microtubule cytoskeleton - also regulate cell cycle progression, transcription, membrane transport - activated by rho-GEFs (60 in human), inactivated by rho-GAPs (70 in human) - inactive rho bound in cytosol to guanine nucleotide dissociating inhibitor (GDI), preventing it to bind to GEF in the membrane Rho, Rac, cdc48...

21. Growth cone extension in axons through the guidance molecules ephrins The most abundant class of protein tyrosin kinases. http://www.youtube.com/watch?v=_1zGQHrvoRo&feature=results_video&playnext=1&list=PLD424D0 006F154019

22. MAP kinase modules - complexes of three protein kinases interconnected in series, often bound to a scaffold - after activation the MAPK-MAPKK interactions may be weakened, enabling MAPK to travel to the nucleus

23. Stress modules MAPKKK MAPKK MAPK Mitogenesis module MAP kinase modules Binding to a scaffold reduces crosstalk BUT also avoids difussion and spreading of signal

24. Signal amplification Signal amplification (of undocked kinases) input signal MEKK1 MKK4 output signal MKK4 JNK modules avoid spreading

25. Downstream of MAPK – the effectors - mostly to promote proliferation and prevent apoptosis - substrates of MAPK: transcription factors, further kinases (MAPKAP kinases), also negative feedback loops through phosphorylation and activation of MAPK phosphatases or ras-GAPs MAPKAP kinases: - MAP kinase activated protein kinases - activation of proliferation and survival programs through phosphorylation of transcription factors or other regulatory proteins - p90 RSK = ribosomal S6 kinase MSK = mitogen and stress activated kinase MNK = MAPK interacting kinase Transctiption factors: - Ets family (Elk-1, TCF... after phosphorylation often bind another TF like SRF) - AP1(homo- or hetero-dimers of c-fos, c-jun, ATF2 and MAF through leucine zipper) - MEF2, MAX, NFAT.....

26. Tyrosin specific phosphatases and GAPs inactivate Ras. Neuronal precursor cell: EGF activation – peaks in 5 minutes, cells later divides NGF activation – lasts many hours, cell stops dividing and differentiates into neurons positive and negative feedback loops, phosphatases Duration of response can influence the final outcome

27. Figure 15-66 Molecular Biology of the Cell (© Garland Science 2008) RTKs GPCRs crosstalk

28. Enzyme-linked receptors 1. receptor tyrosin kinases (RTKs) 2. tyrosin kinases associated receptors 3. receptor serine/threonine kinases 4. histidin kinase associated receptors 5. receptor guanylyl cyclases

29. Similar as receptor tyrosine kinases but the kinase is non-covalently bound to the receptor Tyrosin kinases associated receptors Tyrosin kinases associated receptors Receptors for antigens on leucocytes, for cytokines, hormones, integrin receptors... kinase ligand

30. Tyrosin kinases associated with receptors Src family: Src,Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk - cytoplasmic localization through lipid anchor and through association with receptors - SH2 and SH3 domains - can also be activated by some RTKs and GPCR FAK (focal adhesion kinase) JAK (Janus kinase)

31. Src -first tyrosine kinase discovered, from the chicken Rous sarcoma virus (Peyton Rous, Nobel prize in 1966) = first transmissable agent inducing cancer -oncoproteins are gain of function cellular proteins (Bishop and Varmus,1989) -v-src, c-src

32. FAK (focal adhesion kinase) - associated with integrin receptors in focal adhesion points (places of cell contact with extracellular matrix) - recruit src, mutual phosphorylation and activation of cell survival and proliferation programme

33. JAK-STAT pathway - family of cytokine receptors, receptors for growth hormone and prolactine - stable association with JAK (Janus kinase, JAK1, JAK2, JAK3 and Tyk) - activated JAKs recruits STATs (signal transducers and activators of transcription), in the cytoplasm and phosphorylates them - phosphorylated STATs migrate to the nucleus and activate transcription

34. Enzyme-linked receptors 1. receptor tyrosin kinases (RTKs) 2. tyrosin kinases associated receptors 3. receptor serine/threonine kinases 4. histidin kinase associated receptors 5. receptor guanylyl cyclases

35. TGF-beta / BMP signalling transforming growth factor beta and bone morphogenetic protein

36. - SMADs continuously shuttle between cytoplasm and nucleus during signalling (dephosphorylated in nucleus, rephosphorylated in cytoplasm) - secreted inhibitory proteins regulating the morphogen gradient: noggin and chordin (for BMP), follistatin (for TGFbeta) - inhibitory SMADs (SMAD6 and SMAD7) - compete for activating SMADs - recruits ubiquitin ligase Smurf to the receptor, promoting its internalization and degradation - recruits phosphatases to inactivate the receptor

38. Enzyme-linked receptors 1. receptor tyrosin kinases (RTKs) 2. tyrosin kinases associated receptors 3. receptor serine/threonine kinases 4. histidin kinase associated receptors 5. receptor guanylyl cyclases

39. - bacteria, yeast and plants, not in animals - bacterial chemotaxis: Histidin kinase associated receptors Default movement anticlockwise with occasional tumbling.

40. Chemotaxis receptors Chemotaxis receptors to various atractants and repelents -repellent activates receptor (tumbling), atractant inactivates it (going forward) - histidin kinase CheA (adaptor CheW) - regulator protein CheY binds motor and makes it rotate clockwise - intrinsic phosphatase activity of CheY (accelerated by CheZ)

41. Bacteria senses the difference in the concentrations of repellents and attractants, not the exact concentration!

42. Enzyme-linked receptors 1. receptor tyrosin kinases (RTKs) 2. tyrosin kinases associated receptors 3. receptor serine/threonine kinases 4. histidin kinase associated receptors 5. receptor guanylyl cyclases

43. ANPCR – ANP clearance receptor (endocytosis of ANP) pGC – particulate guanylyl cyclase receptor Natriuretic peptide receptors – activated by ANP hormone produced by atrial cardiomyocytes, regulates blood pressure by relaxing smooth muscles in the veins; also BNP, CNP cGMP Intestinal peptide receptor guanylyl cyclases – activated by heat stable enterotoxin from E.coli and other gut bacteria, stimulates gut secretion and diarrhea; in mammals guanylin and uroguanylin Orphan receptor guanylyl cyclases – no known ligand Receptor guanylyl cyclases

44. NUCLEAR RECEPTORS

45. Figure 15-13 Molecular Biology of the Cell (© Garland Science 2008) NUCLEAR RECEPTORS Ligands: lipophylic molecules Steroid hormones (from cholesterol: sex hormones, vitaminD, cortisol, ecdysone in insects) Thyroid hormones (from tyrosine) Retinoids (from vitaminA)

46. Activation function 1 independent of ligand but very weak, needs to synergize with AF2 Activation function 2 ligand dependent

47. Video tutorial: http://www.nursa.org/flash/gene/nuclearreceptor/start.html I.II. III. IV.

48. Type I nuclear receptors:

49. Type II nuclear receptors:

51. Figure 15-15 Molecular Biology of the Cell (© Garland Science 2008)

52. In vivo ligand sensors

53. STOCHASTICITY IN CELL SIGNALLING

54. Figure 15-24 Molecular Biology of the Cell (© Garland Science 2008) all-or-none effect cell-cell variation cell noise

55. Sprinzak and Elowitz, Nature, 2010 Reporter assay with a GFP reporter Stochastic response

56. ERK2 gene ERK2-YFP fusion in genomic locus YFP Cohen-Saidon C, Alon U et al.: Molecular Cell (2009) Response to EGF

57. Cohen-Saidon C, Alon U et al.: Molecular Cell (2009) Different basal levels… …fold response after EGF stimulation similar between cells ‘Amount of ERK2 entering the nucleus is proportional to the basal level of nuclear ERK2 in each cell, suggesting a fold-change response mechanism.’

58. Estrogen signaling Weber’s law (1834) Human perception of light and sound is not based on absolute levels but on the magnitude of stimulus relative to the background levels.

59. Why fold change rather than absolute level of detection? How is the fold change detected by the cell? It might be easier for the cell to generate reliable fold changes than reliable absolute changes. Negative feed-forward loops. It might serve as an adjustable noise filter. Absolute variations present when background level of stimulus is high tends to be higher than when background level is low.

60. Consequence for regulated gene Z: Pulse of activity Incoherent feed-forward loop as a fold-change detector

61. Incoherent feed-forward loop as a fold-change detector Gene Z serves as a fold change detector.

64. Tranfection assays nucleus GR target Measurable product GR Hormone (cortisol) GRE

65. DBD swapping: ER/GR chimera nucleus GR target Measurable product ER GRE Hormone (cortisol)

68. Ras movie

69. NFkB pathway !!!

70. Ras signaling

71. MAPK signaling

72. FGF signaling

73. Glucocorticoid signaling

74. How to achieve specificity in signalling? 5 MAPK modules in mammals (12 MAPK, 7 MAPKK, 7 MAPKKK...) Both pathways use the same kinase (A) but there is no crosstalk.

75. JNK signaling

76. embryo movie

79. wound healing requires JNK activity

80. MAPK – Ser/Thr specific kinases - activated by dual phosphorylation by MAPKK of a Thr -X - Tyr sequence - rather nonspecific kinase properties in vitro x very specific substrates in vivo. Selectivity improved by docking motifs and scaffold proteins............... MAPK substrate A substrate B MAPK MAPKK – activate MAPK - dual specific kinases = Ser/Thr and Tyr MAPKKK – activated by small GTPases - activate MAPKK by fosforylation of their two Thr or Ser residues - raf for mitogenesis module, MEKK for stress modules (and others). substrate scaffold MAPK... potential phosphorylation sites used phosphorylation site activated by small GTPases and at the same time also by MAPKKKK (page 399 in F.Marks signaling)

81. TGF-beta / activin family and BMP family - transforming growth factor beta and bone morphogenetic protein - during development, they regulate pattern formation and influence various cell behaviors, including proliferation, specification and differentiation, extracellular matrix production, and cell death. In adults - tissue repair, immune regulation,...