Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
G protein–coupled receptor wikipedia , lookup
List of types of proteins wikipedia , lookup
Hedgehog signaling pathway wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
Transcription factor wikipedia , lookup
Cellular differentiation wikipedia , lookup
Signal transduction wikipedia , lookup
Biochemical cascade wikipedia , lookup
Cell Biology - Models The NF-kB/IkB System Yurochko February 19-20, 2008 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 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) NF-kB Biological Implications: Health NF-kB regulation is essential to many aspects of our health including: cellular development cellular survival the immune system What Are We Talking About?? Quick Overview of the NF-kB/IkB Signaling Pathway Simple (?) Model Of The Known Players In NF-kB Induction IL-1 Virus TNF PMA IL-1R1 TNFR1 Other IRAK ? Ras TRADD Cell Memb. PKC TRAF2 TRAF6 NIK MEKK1 MAPK IKK ( and ) pp90rsk IkB Phosphorylation NF-kB/IkB IkB Degradation Free NF-kB NF-kB Translocation NF-kB Responsive Genes Nuclear Memb. The Players NF-kB The IkBs The IKKs Other Upstream Regulators 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. What is NF-kB NF-kB is a heterodimeric transcription factor from the rel-family of transcription factors. Classic NF-kB 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. The NF-kB and IkB Family Schematic of the NF-kB/IkB families Details KEY features QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107. 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 relhomology domain. 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. Picture of Rel-Proteins View of NF-kB binding DNA. NF-kB DNA Transcriptional Regulation by NF-kB -- Mechanisms Binds to a unique sequence found in the kBresponsive 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-kB interacts with HMG-I, bZIP proteins, Sp1, C/EBP c-Rel and RelA interact with TBP RelA interacts with TFIIB Specificity - Transcription Factor Are there other mechanisms of specificity????????? From Science, 2004, 306:632-635 Reminder: How a TF works http://life.nthu.edu.tw/~lslpc/StrucBio/chapter9/chapter9_2.html Reminder: How a TF works Enhancer NF-kB IID TATA BOX -25 Diagram based on and adapted from Struhl, K., Cell 84: 179-182 Initiator Genes Regulated by NF-kB 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 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. Mechanism: NF-kB Activation NF-kB activity is regulated by a family of inhibitors termed IkB which include IkB, IkB, IkB, the p105 and p100 precursors, and Bcl-3. Specifically, the IkBs binds to NF-kB 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-kB from IkB allowing it to translocate to the nucleus and transactivate kBresponsive elements. 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 The NF-kB and IkB Family Schematic of the NF-kB/IkB families Details KEY features QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107. 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. IkB vs. IkB IkB is a 46 kDa protein. First discovered in 1995. Blocks the NLS of NF-kB. Regulates the persistent release of NFkB. Also appears to protect NF-kB from the negative effects of IkB Specificty???? 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. 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 Is IkB really Important???? IkB KO -- born normally but die of a wasting disease by day 7. 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, IKKg (NEMO), is a regulatory subunit (48 kDa). 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). IKKgdoes 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. Schematic of the IKKs Häcker and Karin, 2006, Sci. STKE, 357:1-19. www.stke.org/cgi/contents/full/2006/357/re13 IKK Mechanisms of Action A model of how IKK activity is regulated (both up- and downregulated). Controlled by phosphorylation (kinase dependent event). Häcker and Karin, 2006, Sci. STKE, 357:1-19. www.stke.org/cgi/contents/full/2006/357/re13 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). IKKg KO -- embryonic lethality (similar to the p65 & IKK KO animal). Suggests what??????????? Defects in IKK KOs Hu et al., 1999, Science 284:316-320 More Defects in IKK KOs Hu et al., 1999, Science 284:316-320 Defects in IKK KOs Li et al., 1999, Science 284:321-325 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. Incontinentia Pigmenti Rare familial X-linked dominant condition (Xlinked 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) Affected Males IKKg/NEMO - Another Role IKKg or NEMO can function as a bridge to the interferon signaling pathway IKKg also has the capacity to regukate signal transduction pathways independent of its role in the regulation of NF-kB activation Thus At this point, what do we know? 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 More Details Possible Specificity???? NIK seems to preferentially activate IKK. MEKK1 seems to preferentially activate IKK. Suggests what???? What Activates NF-kB????? Cytokines Growth Factors Cell Adhesion Viral Infection Thus a Receptor-Ligand mediated event. One Last Concluding Figure Molecular Cell Biology; 4th Edition Other mechanisms of specificity????? Everything you ever learned in one cartoon! IMAGES FROM: G. Orphnides and D. Reinberg 2002, Cell 108: 439-451 NF-kB Biological Implications: Health & Human Disease NF-kB regulation is essential to many aspects of our health including: Diseases associated with a dysregulation of NF-kB. cellular development cellular survival the immune system 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. NF-kB Biological Implications: Health NF-kB regulation is essential to many aspects of our health including: cellular development cellular survival the immune system