1. Cell Biology - Models The NF-  B/I  B System Yurochko February 19-20, 2008

2. Lecture Goal & Outline  Goal:  To introduce you to a model of signal transduction and specifically examine a signaling pathway. The pathway being the NF-  B/I  B regulatory pathway.  Outline:  NF-  B  I  Bs  IKKs  Upstream Regulators

3. NF-  B Biological Implications: Human Disease  Diseases associated with a dysregulation of NF-  B.  Atherosclerosis  Asthma  Arthritis  Cancer  Diabetes  Inflammatory bowl disease  Stroke  Viral Infections (AIDS)

4. NF-  B Biological Implications: Health  NF-  B regulation is essential to many aspects of our health including:  cellular development  cellular survival  the immune system

5. What Are We Talking About??  Quick Overview of the NF-  B/I  B Signaling Pathway

6. The Players  NF-  B  The I  Bs  The IKKs  Other Upstream Regulators

7. History of NF-  B  Discovered in 1986 in the laboratory of Dr. David Baltimore.  Found as a nuclear factor in B cells.  Found to transactivate the kappa light chain promoter.  Later found to activate many genes.

8. What is NF-  B  NF-  B is a heterodimeric transcription factor from the rel-family of transcription factors.  Classic NF-  B is made up of two subunits termed p50 and p65.  Other members include c-rel, RelB, p52, as well as the two precursors p105 and p100.  Multiple subunits all interact to form a variety of factors with different apparent functions.  Evolutionarily conserved family of proteins.

9. The NF-  B and I  B Family  Schematic of the NF-  B/I  B families  Details KEY features DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.

10. NF-  B as a Transcription Factor  Contains a DNA binding domain and a transactivation domain.  The p65 subunit contains the transactivation domain and the p50 subunit contains the DNA binding domain.  The NF-  B subunits contains a rel- homology domain.

11. RHD - Defines this Family  Common to all members of the NF-  B family.  Is ~ 300 a.a. domain.  Is a multifunctional domain.  Controls NF-  B dimerization.  Allows interaction with the IkBs.  DNA Binding.  Contains the NLS.

12. Picture of Rel-Proteins  View of NF-  B binding DNA. DNA NF-  B

13. Transcriptional Regulation by NF-  B -- Mechanisms  Binds to a unique sequence found in the  B- responsive promoters (5’-GGGRNNYYCC-3’).  c-Rel, RelB, and RelA (p65) contain transactivation domains.  The NF-  B family members interact with other transcription factors and members of the basal transcriptional machinery.  NF-  B interacts with HMG-I, bZIP proteins, Sp1, C/EBP  c-Rel and RelA interact with TBP  RelA interacts with TFIIB

14. Specificity - Transcription Factor Are there other mechanisms of specificity????????? From Science, 2004, 306:632-635

15. Reminder: How a TF works http://life.nthu.edu.tw/~lslpc/StrucBio/chapter9/chapter9_2.html

16. Reminder: How a TF works Diagram based on and adapted from Struhl, K., Cell 84: 179-182 Enhancer Initiator TATA BOX -25 IID NF-  B

17. Genes Regulated by NF-  B Good vs. Bad  The Good  Immune Responsive Genes  Cytokine Genes  Adhesion Molecules  Transcription Factors  Growth Factors and Proliferative Genes  The Bad  Viral Promoters  Growth Factors and Proliferative Genes  Inflammatory Genes

18. Is NF-  B really Important????  Through the use of Knock-Out animals the critical role NF-  B plays in health has been demonstrated.  p65 KO -- embryonic lethality.  p50 KO -- develops normally, but has B cell immune defects.  RelB KO -- develops normally, but has immune defects and changes in hematopoiesis.  c-rel KO -- develops normally, but B cells and T cells are unresponsive to certain activating signals.

19. Mechanism: NF-  B Activation  NF-  B activity is regulated by a family of inhibitors termed I  B which include I  B , I  B , I  B , the p105 and p100 precursors, and Bcl-3.  Specifically, the I  Bs binds to NF-  B and keeps it sequestered in an inactive state in the cytosol.  Following cellular activation (by many different stimuli (cytokines, mitogens, viral infection, etc.), a complex signaling cascade is initiated which ultimately frees NF-  B from I  B allowing it to translocate to the nucleus and transactivate  B- responsive elements.

20. The I  Bs  There are two main I  Bs  I  B   I  B   There are also other less studied I  Bs or I  B like molecules.  I  B   The C-terminal portions of p100 and p105.  BCL3

21. The NF-  B and I  B Family  Schematic of the NF-  B/I  B families  Details KEY features DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.

22. I  B  vs. I  B   I  B  is the prototypic I  B.  We first discovered it in 1990.  It is a 37 kDa protein.  Binds to NF-  B and blocks its NLS.  Regulates the rapid release of NF-  B and its rapid down regulation.  Also contains a nuclear export signal which is important in the removal of NF-  B from the nucleus.

23. I  B  vs. I  B   I  B  is a 46 kDa protein.  First discovered in 1995.  Blocks the NLS of NF-  B.  Regulates the persistent release of NF-  B.  Also appears to protect NF-  B from the negative effects of I  B   Specificty????

24. Mechanism: I  B Regulation  The I  Bs contain critical serine residues.  I  B  -- S32 & S36  I  B  -- S19 & S23  These serines are the targets of upstream serine kinases termed IKKs (I  B Kinases).  Following phosphorylation, the I  Bs are ubiquitinated and targeted for degradation by the 26S proteasome.

25. I  B Degradation  Schematic of the regulatory serines and a quick look at the ubiquitination event (occurs at lysines 21 and 22 on I  B  ).  E1 - ubiquitin-activating enzyme  E2 - ubiquitin-conjugating enzyme  E3 - ubiquitin-ligating enzyme

26. Is I  B really Important????  I  B  KO -- born normally but die of a wasting disease by day 7.

27. The IKKs  There is an IKK complex composed of three known subunits. May include others, as the complex is 700-900 kDa.  Two of the members, IKK  and IKK  are catlytic subunits (85 & 87 kDa, respectively).  The third member, IKK  (NEMO), is a regulatory subunit (48 kDa).

28. The IKKs  IKK  and IKK  have a very similar primary structure (52% a.a. identity, ~70% DNA identity). Contain the same domains.  a leucine zipper (for protein-protein interactions),  a helix-loop-helix domain (regulatory function),  a kinase domain (functional properties).  IKK  does not contain a catalytic domain and is very different from IKK  and IKK . Probably interacts with IKK  and IKK  as a dimer or a trimer.

29. Schematic of the IKKs Häcker and Karin, 2006, Sci. STKE, 357:1-19. www.stke.org/cgi/contents/full/2006/357/re13

30. IKK Mechanisms of Action  A model of how IKK activity is regulated (both up- and down- regulated).  Controlled by phosphorylation (kinase dependent event). Häcker and Karin, 2006, Sci. STKE, 357:1-19. www.stke.org/cgi/contents/full/2006/357/re13

31. Are the IKKs really Important?  In Mice:  IKK  KO -- born alive but died shortly after birth. Showed severe muscular and skeletal defects. Had normal activation of NF-  B following proinflammatory stimuli.  IKK  KO -- embryonic lethality (similar to the p65 KO animal).  IKK  KO -- embryonic lethality (similar to the p65 & IKK  KO animal).  Suggests what???????????

32. Defects in IKK  KOs Hu et al., 1999, Science 284:316-320

33. More Defects in IKK  KOs Hu et al., 1999, Science 284:316-320

34. Defects in IKK  KOs Li et al., 1999, Science 284:321-325

35. Are the IKKs really Important?  In humans, there is a diagnosed genetic defect in which IKK  is absent. (Called Incontinentia Pigmenti)  In males - embryonic lethality (usually)  In females -- congenital disorder of teeth, hair, and sweat glands, death usually occurs early in life.

36. Incontinentia Pigmenti  Rare familial X-linked dominant condition (X- linked recessive trait (chromosomal locus Xq28)).  Characteristics include  Skin lesions  Hair, eye, teeth, and nail abnormalities  Osteosclerosis  Immune system disorders (immunodeficiency resulting in recurrent infections)  Some males do survive for several years (usually have a milder genetic abnormality)

37. Affected Males

38. IKK  /NEMO - Another Role  IKK  or NEMO can function as a bridge to the interferon signaling pathway  Thus IKK  also has the capacity to regukate signal transduction pathways independent of its role in the regulation of NF-  B activation

39. At this point, what do we know?

40. Other Upstream Regulators  The are many upstream regulators described in the literature. How each upstream kinase fits in, is unclear, especially in regards to specific signaling.  IKK regulation appears to be a point of convergence for a number of different signaling pathways.  Some of the upstream players include:  NIK (NF-  B Inducing Kinase)  MEKK1 (A MAP3K)  Ras/Raf  Others

41. More Details Possible Specificity????  NIK seems to preferentially activate IKK .  MEKK1 seems to preferentially activate IKK .  Suggests what????

42. What Activates NF-  B?????  Cytokines  Growth Factors  Cell Adhesion  Viral Infection  Thus a Receptor-Ligand mediated event.

43. One Last Concluding Figure Other mechanisms of specificity????? Molecular Cell Biology; 4th Edition

44. Everything you ever learned in one cartoon! IMAGES FROM: G. Orphnides and D. Reinberg 2002, Cell 108: 439-451

45. NF-  B Biological Implications: Health & Human Disease  NF-  B regulation is essential to many aspects of our health including:  cellular development  cellular survival  the immune system  Diseases associated with a dysregulation of NF-  B.  Atherosclerosis, Asthma, Arthritis, Cancer, Diabetes, Inflammatory bowl disease, Stroke, Viral Infections (AIDS)  Thus, together this is a critical pathway and one that warrants much attention to understand its role in human pathobiology.

46. NF-  B Biological Implications: Health  NF-  B regulation is essential to many aspects of our health including:  cellular development  cellular survival  the immune system