Cell Biology - Models The NF- k B/I k B System

1. Cell Biology - ModelsThe NF-kB/IkB 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-kB/IkB regulatory pathway. • Outline: • NF-kB • IkBs • IKKs • Upstream Regulators

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

4. NF-kB Biological Implications:Health • NF-kB 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-kB/IkB Signaling Pathway

6. The Players • NF-kB • The IkBs • The IKKs • Other Upstream Regulators

7. History of NF-kB • 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-kB • 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-kB and IkB Family • Schematic of the NF-kB/IkB families • Details KEY features DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.

10. NF-kB 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-kB subunits contains a rel-homology domain.

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

12. Picture of Rel-Proteins • View of NF-kB binding DNA. NF-kB DNA

13. Transcriptional Regulation by NF-B -- Mechanisms • Binds to a unique sequence found in the kB-responsive promoters (5’-GGGRNNYYCC-3’). • c-Rel, RelB, and RelA (p65) contain transactivation domains. • The NF-kB 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. Enhancer IID Initiator TATA BOX -25 Reminder: How a TF works NF-B Diagram based on and adapted from Struhl, K., Cell 84: 179-182

17. Genes Regulated by NF-BGood 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-kB really Important???? • Through the use of Knock-Out animals the critical role NF-kB 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-kB 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 IkBs • There are two main IkBs • IkB • IkB • There are also other less studied IkBs or IkB like molecules. • IkB • The C-terminal portions of p100 and p105. • BCL3

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

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

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

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

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

26. Is IkB really Important???? • IkB 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, IKKa and IKKb are catlytic subunits (85 & 87 kDa, respectively). • The third member, IKKg (NEMO), is a regulatory subunit (48 kDa).

28. The IKKs • IKKa 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 IKKa 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-kB 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 IKKa KOs Hu et al., 1999, Science 284:316-320

33. More Defects in IKKa 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 IKKg 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. IKKg/NEMO - Another Role • IKKg or NEMO can function as a bridge to the interferon signaling pathway • Thus IKKg 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-kB Inducing Kinase) • MEKK1 (A MAP3K) • Ras/Raf • Others

41. More Details Possible Specificity???? • NIK seems to preferentially activate IKKa. • MEKK1 seems to preferentially activate IKKb. • Suggests what????

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

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

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

45. NF-kB Biological Implications:Health & Human Disease • NF-kB regulation is essential to many aspects of our health including: • cellular development • cellular survival • the immune system • Diseases associated with a dysregulation of NF-kB. • 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-kB Biological Implications:Health • NF-kB regulation is essential to many aspects of our health including: • cellular development • cellular survival • the immune system