1. 1 Molecular Biology of Cancer Signal Transduction 2

2. 2 Molecular Biology of Cancer 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

3. 3 Molecular Biology of Cancer 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

4. 4 Molecular Biology of Cancer 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

5. 5 Molecular Biology of Cancer Ras (from rat sarcoma) is a GEF guanine nucleotide exchange factor GTPase activity

6. 6 Molecular Biology of Cancer GDP  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 proline- rich sequence in the C-terminus of SOS (a guanine nucleotide exchange factor)  SOS is recruited to the close proximity of RAS in the membrane  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 P P P P SH2 SH3 GRB2 SOS RAF PGTP 14-3-3 RAS

7. 7 Molecular Biology of Cancer

8. 8 MAPK  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.  Activated MAPK:  phosphorylates a number of substrates in the plasma membrane and the cytoplasm;  it is also translocated into the nucleus (within minutes) where it phosphorylates nuclear transcription factors.  Transcription of genes important for cell proliferation.GDP P P P P SH2 SH3 GRB2 SOS RAF P GTP 14-3-3 RAS MEK P P PP Substrates P P P MAPK PP

9. 9 Molecular Biology of Cancer Homework Write no more that 2 papers about the regulation of Raf activity.

10. 10 Molecular Biology of Cancer 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

11. 11 Molecular Biology of Cancer The MAPK signalling pathways  It should be noted that the RAS  RAF  MEK  MAPK 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  JNK pathway:  a family of MAPK relatives known as JNKs (also called stress- activated protein kinase (SAPKs)  become activated in response to extracellular stresses  like cycloheximide treatment, UV irradiation, heat shock, or TNF-  treatment,.

12. 12 Molecular Biology of Cancer RAC1/CDC42 GTP STRESS PAK MEKK1-3 P P MEK4 P P  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.  GTP-bound form of RAC1 and CDC42 bind and activate the serine/threonine protein kinase PAK, PKN, and PtdIns kinases.  these kinases phosphorylate and activate MEKK1-3  MEKK1-3 phosphorylate and activate MEK4 (also called JNKK) (~ MEK in MAPK pathway; 45% identical in sequence with MEK) PAK: p21-activated protein kinase PKN: protein kinase N PtdIns kinase: phosphatidylinositol kinase

13. 13 Molecular Biology of Cancer RAC1/CDC42 GTP STRESS PAK MEKK1-3 P P MEK4 P P JNK P P c-JUN P JNK P P  MEK4 phosphorylates JNK at two similar sites as in ERK (but T-P-Y in JNK instead of T-E-Y in MAPK)  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 N- terminal residues (Ser63 and Ser73)  activation of transcription by c- JUN

14. 14 Molecular Biology of Cancer RAC1/CDC42 GTP STRESS PAK MEKK1-3 P P 14-3-3 P P P P SH2 SH3 GRB2 SOS RAS GTP RAF MEK4 P P MEK P P JNK P P MAPK PP

15. 15 Molecular Biology of Cancer Generic pathway MAPKKK MAPKK MAPK ERK/MAP kinase pathway RAF1 MEK1,2 ERK1,2 RAS GRB2/SOS Receptor PTK JNK/SAPK pathway RAC/CDC42 MEKK1-3 MEK4 JNK/SAPK TAK MEK3,6 p38 pathway PAK Stress responsesProliferation/differentiation

16. 16 Molecular Biology of Cancer 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?

17. 17 Molecular Biology of Cancer Specificity of MAP kinase pathways  One way is by scaffold proteins.

18. 18 Molecular Biology of Cancer 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

19. 19 Molecular Biology of Cancer G Protein-Linked Receptors  A number of different hormones mediate biological responses by binding to G protein-linked receptors   - and  -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

20. 20 Molecular Biology of Cancer G Protein  G proteins are guanine nucleotide-binding proteins composed of  -,  -, and  -subunits  The  -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  -subunit binds to the guanine nucleotide (GDP or GTP)  So far we have identified a G s (  s ), a G i (  i ), a G q (  q ) and a G t (  t ) protein Distinct from the monomeric GTP- binding proteins GTPase e.g. Ras

21. 21 Molecular Biology of Cancer

22. 22 Molecular Biology of Cancer G Protein-Linked Receptors  Seven-spanning G protein-linked receptors:  contain seven stretches of ~22-24 hydrophobic residues, forming seven transmembrane  helices  G protein binds to: 1.the loop between  helices 5 and 6; and 2.the C-terminal region

23. 23 Molecular Biology of Cancer 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.

24. 24 Molecular Biology of Cancer Example of G protein-linked receptor Adrenaline receptor 1.Hormone binding to  - 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).

25. 25 Molecular Biology of Cancer  Binding of hormone to  -adrenergic receptors  conformational change in loop between helices 5 and 6  bind to G s in such a way that GDP is displaced and GTP is bound  G  and G  are dissociated from G s  -GTP  G s  -GTP is able to bind to and activate adenylate cyclase  activated adenylate cyclase can then produce cAMP from ATP  GTP bound to G s  is quickly hydrolysed to GDP (seconds)  association of G  and G  with G s  -GDP  inactivation of adenylate cyclase GG GbGb G Sa GDP AC GG GbGb G Sa GDP GTP GTP ATP cAMP + PP i G Sa GTPGDP

26. 26 Molecular Biology of Cancer Amplification of signal 1.Activated G s  -GTP can diffuse rapidly   one activated receptor can activate many G s. 2.One G s  -GTP can bind to only one adenylate cyclase  but this can catalyze the synthesis of many cAMP.

27. 27 Molecular Biology of Cancer 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 G s  (at Arg174, which is located near the GTP-binding site in G s  )  modified G s  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.

28. 28 Molecular Biology of Cancer  G i may inhibit adenylate cyclase by two mechanisms: 1.The  i -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  s -subunits preventing activation of adenylyl cyclase

29. 29 Molecular Biology of Cancer 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.

30. 30 Molecular Biology of Cancer 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  -subunit gene  Preprosomatostatin  Vasoactive intestinal polypeptide  A surfactant protein, SP-A  Several isoforms of cytochrome P450

31. 31 Molecular Biology of Cancer 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.

32. 32 Molecular Biology of Cancer The G q protein-linked receptors and Ca 2+  Ca 2+ is an important intracellular second messenger.  [Ca 2+ ] in the cytosol is low (10 -7 M)  [Ca 2+ ] outside the cell is high (10 -3 M)  [Ca 2+ ] in ER also high.  Extracellular signals open Ca 2+ channels in plasma / ER membranes  Ca 2+ rushes into the cytosol  increase Ca 2+  Ca 2+ - dependent responses.

33. 33 Molecular Biology of Cancer Control of cytosolic calcium  Ca 2+ -ATPase in plasma membrane and ER membrane pumps Ca 2+ out of the cytosol (use ATP as energy) into the extracellular space and the ER respectively.  Normally, free [Ca 2+ ] changed from ~10 -7 M in resting cells to ~5x10 -6 M in stimulated cells.  If Ca 2+ pumps are defective and the free [Ca 2+ ] in the cytosol gets to >10 -5 M, a low affinity, high capacity Ca 2+ pump in the inner mitochondrial membranes kicks in and pump Ca 2+ into the mitochondria (uses electrochemical gradient as energy).

34. 34 Molecular Biology of Cancer Adapted from Molecular Biology of the Cell

35. 35 Molecular Biology of Cancer GG GG GqGq GDP PLC-  IP 3 :Inositol triphosphate Release Ca 2+ from ER DAG: Diacylglycerol Activates PKC Overview 1.Extracellular signaling molecules binds to G protein-linked receptor in the plasma membrane.  Activation of a G protein G q 2.Activation of phospholipase C-  3.Cleaves phosphatidylinositol bisphosphate (PIP 2 ) into two products:  2 different signal transduction pathways

36. 36 Molecular Biology of Cancer Phosphatidylinositol (PI) is a minor phospholipid in cell membranes; PIP 2 is a phosphorylated derivative of PI - located in the inner half of the plasma membrane lipid bilayer.

37. 37 Molecular Biology of Cancer

38. 38 Molecular Biology of Cancer IP 3 activates Ca 2+ release from the ER  IP 3 binds to the IP 3 -gated Ca 2+ release channels in the ER membrane  release Ca 2+ into the cytosol (by gradient).  Depleted Ca 2+ store promotes influx of extracellular Ca 2+ via membrane channels (signals by the release Ca 2+ or factor from empty store?)

39. 39 Molecular Biology of Cancer IP 3 DAG ER IP 3 -gated Ca 2+ release channels Ca 2+ Calmodulin 4 high-affinity Ca 2+ -binding sites Enzymes (e.g. myosin light-chain kinase, phosphorylase kinase, Ca 2+ -calmodulin kinase II etc) Membrane transport proteins (e.g. Ca 2+ -ATPase on plasma membrane

40. 40 Molecular Biology of Cancer 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 Ca 2+ are mediated by Ca 2+ /calmodulin-dependent kinases (CaM- kinases).  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.

41. 41 Molecular Biology of Cancer

42. 42 Molecular Biology of Cancer CaM kinases  Functions of CaM-kinase II: Molecular memory device  switching to active state when exposed to Ca 2+ /calmodulin.  remains active by autophosphorylation (i.e. remains active even when Ca 2+ 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)

43. 43 Molecular Biology of Cancer

44. 44 Molecular Biology of Cancer Termination of Ca 2+ response 1.Breakdown of DAG 2.Further phosphorylation of PIP 2 3.IP 3 is dephosphorylated and inactivated by phosphatases. (sometimes it is further phosphorylated to IP 4 to mediate other responses) 4.Ca 2+ is pumped out of the cell by Ca 2+ -ATPase 5.Phosphatases which inactivate CaM-kinase II

45. 45 Molecular Biology of Cancer 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 Ca 2+ activates PKC (a seine/threonine kinase)  DAG increases the affinity of PKC for Ca 2+ and for phospholipids  Phospholipid and Ca 2+ binding activate PKC which phosphorylates serine and threonine residues of certain cellular proteins

46. 46 Molecular Biology of Cancer IP 3 DG PKC  Ca 2+ induces PKC to move from cytosol to plasma membrane  PKC is activated by Ca 2+, DG (and a membrane phospholipid phosphatidylserine) at the plasma membrane  Activated PKC then phosphorylates several substrates ABC PP P

47. 47 Molecular Biology of Cancer 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 c- Jun (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

48. 48 Molecular Biology of Cancer MAPK IkB NFkB Gene 1 Gene 2 DG PKC P P Nucleus P Activates transcription

49. 49 Molecular Biology of Cancer Receptor crosstalk

50. 50 Molecular Biology of Cancer Receptor-linked Tyr kinases This is a common motif. It is called the Jak/STAT pathway for gene regulation.

51. 51 Molecular Biology of Cancer Signal transduction by nuclear receptors

52. 52 Molecular Biology of Cancer e.g. Glucocorticoid receptor response element: 5’-AGAACA(N) 3 TGTTCT-3’ 3’-TCTTGT(N) 3 ACAAGA-5’ 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).

53. 53 Molecular Biology of Cancer Glucocorticoid receptor - a C4 zinc-finger homodimer

54. 54 Molecular Biology of Cancer N C 123 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

55. 55 Molecular Biology of Cancer Hormone BD DNA BD AD GLU Hormone BD DNA BD AD EST

56. 56 Molecular Biology of Cancer Hormone BD DNA BD AD GLU  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.

57. 57 Molecular Biology of Cancer Inactive w/o hormone 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 DNA BD AD Release inhibition DNA BD AD

58. 58 Molecular Biology of Cancer 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

59. 59 Molecular Biology of Cancer  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 Hormone BD DNA BD AD NLS Hsp90 GLU Nuclear membrane