Molecular and Cellular Pharmacology Student Seminars 2002-2003

Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain.

Sakurada K, Ohshima-Sakurada M, Palmer TD, Gage FH.
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

Adult rat-derived hippocampal progenitor cells express many of the molecules implicated in midbrain dopaminergic determination, including FGF receptors 1, 2 and 3, the sonic hedgehog receptor components Smo and Ptc, and the region-specific transcription factors Ptx3 and Nurr1. Here we use undifferentiated progenitors to probe the events leading to the dopaminergic phenotype and find that the influences of Nurr1 can be temporally and mechanistically uncoupled from the patterning influences of sonic hedgehog and FGF-8 or the more generic process of neuronal differentiation itself. In gain-of function experiments, Nurr1 is able to activate transcription of the tyrosine hydroxylase gene by binding a response element within a region of the tyrosine hydroxylase promoter necessary for midbrain-specific expression. This activation is mediated through a retinoid X receptor independent mechanism and occurs in all precursors, regardless of differentiation status. Overexpression of Nurr1 does not affect proliferation or stimulate neuronal differentiation and has no influence on the expression of other dopaminergic markers. This uncoupling of tyrosine hydroxylase expression from other dopaminergic markers suggests that the midbrain dopaminergic identity is dictated by a combination of pan-dopaminergic (e.g., Shh/FGF-8) and region-specific (Nurr1) mechanisms.

Reference
Development 1999 Sep;126(18):4017-26

Young Cho
Pharmacology 901 Student Seminar
Monday, September 30, 2002
12:00 Noon
Genetics Building - 1111 Auditorium
425 Henry Mall

AhR Activity is Regulated by Kinase Pathways in Suspension Condition

The aryl hydrocarbon receptor (AhR) is activated by planar polycyclic ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through interaction with the PAS-B domain. AhR activation in the absence of an exogenous ligand occurs when keratinocytes and hepatoma cells are cultured in methylcellulose suspension. Here, we have characterized AhR activation in C3H10T¬®¯ö mouse embryonic fibroblast (MEF) clonal sub-lines engineered to contain two luciferase reporters, which respond to AhR activation. In most sub-lines carrying internal AhR-responsive reporter, induction of the internal reporters, which was mediated by AhR activation in suspension culture, paralleled a comparable TCDD stimulation in confluent monolayer culture. Suspension activation alone declined substantially within 24 hrs. For a subset of lines, suspension activation of either reporter exceeded TCDD-induced activation by over two fold, even though these activations were comparable for the Cyp1B1 mRNA in these lines. Two other activation procedures (methylcellulose overlay and low density cultures) showed similar stimulations while retaining cell adhesion. AhR activation by suspension, overlay monolayer, or low-density monolayer culture was completely insensitive to a competitive TCDD antagonist, a-naphthoflavone (aNF). In contrast, AhR activation by TCDD/monolayer or suspension was clearly inhibited by 3-MNF, a TCDD antagonist through prevention of nuclear translocation of AhR. AhR activation is, therefore, not due to a constitutive TCDD-like endogenous ligand. AhR activation was essentially blocked when cell-cell contacts were restored indicating cell-cell contacts are rather critical for AhR activity. Each TCDD/monolayer and suspension activation produced a similar nuclear co-translocation of AhR with Hsp90 and inhibition by the geldanamycin, an Hsp90 ATPase inhibitor. Nuclear translocation and turnover of AhR was also similarly regulated in both TCDD/monolayer and suspension processes. Nevertheless, suspension activation was clearly distinguished through substantial further activation by LiCl, a selective GSK-3 inhibitor. Suspension activation and potentiation by lithium were completely reversed by Ro31-8220, a selective PKC inhibitor, but not by the structurally similar inhibitor, Bisindoylmaleimide I. These reagents did not affect nuclear translocation and turnover of the AhR. The endogenous Cyp1B1 gene showed similar response characteristics. We suggest that the ligand-independent process in suspension activation involves a distinct co-activation process that is sensitive to protein kinase signaling pathway on which both Li and Ro31 act.

Jianlin Chu
Monday, October 7, 2002
Fluno Center

Repression of AP-1-mediated Transactivation by the Notch-1 Intracellular Domain

Developmental decisions that control cell fate are commonly regulated by the Notch signaling pathway. In the canonical Notch pathway, signal transduction is initiated when the extracellular domain of Notch binds to its ligand on adjacent cells, resulting in activation of the intracellular domain of Notch (NIC). NIC translocates to the nucleus and binds to a transcriptional repressor (CSL, CBF1/Su(H)/Lag-1), converting it into an activator, thereby activating target genes. We found that in addition to mediating CSL-dependent activation, the intracellular domain of human Notch-1 (NIC-1) represses AP-1-mediated transactivation [Chu J. et al. (2002) J. Biol. Chem. 277: 7587 7597]. As numerous genes that control immune and inflammatory responses are AP-1-dependent, and Notch regulates immune cell function, we investigated the underlying molecular mechanisms. Repression correlated with predominant nuclear localization of NIC-1 and was not accompanied by disruption of c-Jun N-terminal kinase-dependent signaling events required for AP-1 activation nor defective AP-1 DNA binding activity. Surprisingly, the RAM domain of NIC-1, whose only function was to mediate NIC-CSL binding, is also required for repression of AP-1. To further investigate the sequence requirements for CSL activation and AP-1 repression, we generated a series of RAM domain internal deletion mutants and found that two thirds of the RAM domain from the N-terminus is crucial for both activation and repression. Deletions within the last 40 amino acids of this region have little impact on activation and repression, implicating the first 20 amino acids in conferring activation and repression. Analysis of this minimal region should yield important information about sequence requirements for CSL-dependent activation and AP-1 repression. This information will help determine whether activation and repression involve distinct protein-protein interactions or if a single interactor mediates both activities. Based on the importance of AP-1 target genes in hematopoiesis, vasculogenesis, and various immune cell functions, crosstalk between Notch and AP 1 pathways is likely to have critical roles in the control of these processes.

Tony T. Huang
Monday, October 7, 2002
Fluno Center

A defect in the NF-kB signaling pathway: How do mutations in the NEMO zinc-finger domain affect DNA damage-dependent activation of NF-kB?

Activation pathways of the transcription factor NF-kB typically originate from cytokine- or lipopolysaccharide (LPS)-induced ligand-receptor interactions on the cell membrane. However, NF kB can also be activated by a group of agents that damage DNA in the nucleus. The DNA damage dependent NF-kB signaling pathway appears to require downstream molecular components and events similar to that of the cell membrane receptor-induced cannonical signaling cascade. For instance, DNA damage-induced activation of NF-kB, like the canonical pathway, involves the activation of the IkB kinase complex (IKK), IKK-dependent phosphorylation of the NF-kB inhibitor protein, IkBa, ubiquitination and degradation of IkBa by the 26S proteasome, resulting in the release of NF-kB to the nucleus to activate target gene transcription. However, the mechanism by which IKK activity in vivo is induced by different upstream signaling pathways is unclear. Here, we will present some of our preliminary observations that the putative zinc-finger domain in the regulatory subunit of the IKK complex, NEMO (IKKg), is indeed critical for the DNA-damage dependent activation of IKK and NF-kB. In contrast, the disruption of the NEMO zinc-finger domain appears to be less severe for LPS- and cytokine-induced NF-kB signaling pathways. Interestingly, recent reports show that patients containing mutations in the putative zinc-finger domain of NEMO result in an X-linked primary immunodeficiency characterized by hyper-IgM syndrome and hypohidrotic ectodermal dysplasia (XHM-ED). Thus, the human disease phenotype caused by the disruption of the NEMO zinc-finger domain is due to impaired but not abolished NF-kB signaling in these patients.

Indra Lim
Monday, October 7, 2002
Fluno Center

NMDA receptor interactions with PSD-95: Structural and Functional Implications

Molecular and Cellular Pharmacology Program Post-synaptic Density protein 95 (PSD-95) is a structural protein that binds to NMDA-type glutamate receptors at the post-synaptic site. The essential role of the NMDA receptor in synaptic function, including learning and memory suggests that PSD-95 may be important for these functions. NMDA receptors bind protein-protein interaction domains on PSD-95 called PDZ domains. In order to study how the primary structure of the NMDA-receptor affects affinity for PDZ domains, we constructed several libraries of peptides based on the PSD-95 binding domain of the NMDA receptor subunit NR2b, systematically substituting at each position archetypical amino acids. Using fluorescence anisotropy, we detemined the affinity of the peptides for individual PDZ domains of PSD-95 and SAP102, a related protein. From these data, we concluded that an optimal sequence for binding to the PDZ1 and 2 and PSD-95 and SAP102 followed the template E/Q-S/T-D/E/Q/N-V. Searching the human genome for proteins ending with E/Q-S/T-X-V turned up several proteins. We tested and found several proteins for that had not previously been described to bind PSD-95 and SAP102. We are currently investigating the abilities of selected peptides for their ability to compete with endogenous NR2b/SAP complexes in intact neurons and the physiological consequences of such a competition.

Blue-leaf Hannah
Monday, October 7, 2002
Fluno Center

Structural Characterization of a Thrombospondin-1 Polymorphism Associated with Premature Familial Coronary Heart Disease

A single nucleotide polymorphism (SNP), which substitutes a serine for an asparagine at residue 700 in the Ca2+-binding repeats of thrombospondin-1 (TSP-1), is associated with premature familial coronary heart disease. Missense mutations that localize to a homologous region of Cartilage Oligomeric Matrix Protein (COMP, TSP-5) result in skeletal dysplasias. We hypothesized that, similar to COMP mutations, the S700 polymorphism may be associated with altered conformation of the Ca2+-binding repeats of TSP-1. Using bacculoviruses, we expressed the Ca2+-binding repeats alone (Ca-1) and the Ca2+-binding repeats with the third EGF repeat (E3Ca-1), without (N700) and with (S700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (W698), adjacent to the polymorphic residue, was strongly and cooperatively perturbed by Ca2+. The third EGF repeat dramatically altered the Ca2+ ion dependence of the fluorescence transition for the N700 constructs: EC50 of Ca-1 N700 = 400_M ± 40, Hill Coefficient (nH) =3.4 ± 0.2; EC50 of E3Ca-1 N700 = 70_M ± 3, nH = 5.5 ± 0.4). The S700 polymorphism shifted the EC50 to higher calcium concentrations: EC50 of Ca-1 S700 = 960_M ± 20, nH= 2.2 ± 0.1; EC50 of E3Ca-1 S700 = 110_M ± 4, nH = 7.1 ± 0.5. This destabilizing effect is due to local conformational changes, as the S700 polymorphism did not greatly influence the secondary structure of E3Ca-1 or Ca-1 as assessed by far UV CD. At 200_M Ca2+, in which both E3Ca-1 N700 and S700 are in the Ca2+-replete conformation, E3Ca-1 S700 was completely destabilized at 50*C compared to 65*C for E3Ca-1 N700 when measured by fluorescence. These findings indicate that the S700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.

Renee L. Doughman
Monday, October 7, 2002
Fluno Center

The type Ig phosphatidylinositol phosphate kinase targets to and regulates focal adhesions.

The ability of cells to form cell contacts, adhere to the extracellular matrix, change morphology, and migrate are events essential for development, wound healing, metastasis, cell survival, and the immune response. These events are dependent upon integrin binding to the extracellular matrix and assembly of focal adhesions. Focal adhesions are complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. Phosphatidylinositol-4,5 bisphosphate (PI4,5P2) regulates interactions between these proteins, including the interaction of vinculin with actin and talin. The binding of talin to b-integrin is strengthened by PI4,5P2, suggesting that the basis of focal adhesion assembly is regulated by this lipid mediator. Here we show that the type I phosphatidylinositol phosphate kinase gamma (PIPKIg661), an enzyme that makes PI4,5P2, is targeted to focal adhesions by an association with talin. PIPKIg661 is tyrosine phosphorylated by focal adhesion associated kinase (FAK) signaling, increasing both PIP kinase activity and association with talin. This defines a mechanism for spatial generation of PI4,5P2 at focal adhesions.

Pei-Yun Beth Chang
Miyamoto Lab
Pharmacology 901 Student Seminar
Monday, October 14, 2002
12:00 Noon
Genetics Building - 1111 Auditorium
425 Henry Mall

NF-kB Super-Induction: A Mechanistic Investigation on the Novel Cross-talk Pathway for NF-kB Signaling

The NF-kB/Rel transcription factor family comprises a group of conserved proteins that play pivotal roles in various physiological as well as developmental processes. NF-kB regulates transcription of inflammatory genes including the cytokines, leukocyte adhesion molecules and inflammatory enzymes and anti-apoptotic genes that promote survival of a variety of cell types. Deregulation of the NF-kB signaling cascade is therefore implicated in the pathogenesis of diseases involved in dysfunctions of the inflammation and apoptotic response, including atherosclerosis, autoimmune diseases, lung fibrosis and carcinogenesis. Interestingly, various DNA damaging agents that are used to treat cancer can induce NF-kB activation, which potentially underlies a mechanism of resistance for cancer treatment. The long term goal of the proposed research is to develop strategies that exploit NF-kB signaling to control the pathogenesis of carcinogenesis.

We discovered that NF-kB activation by several DNA double strand inducing agents is augmented by prior treatment of TPA (12-myristate-13-acetate). This response was observed in several human and rodent cell types, suggesting that it is a highly conserved crosstalk pathway. The underlying mechanism and physiological importance of the crosstalk of the two ubiquitious signaling cascades have not been investigated. I will attempt to determine the mechanism of "super-induction" and begin to investigate its role by focusing on cell survival regulation in several cell types.

1) To determine the molecular mechanism through which PKC super-induces NF-kB activation, we will ask the following questions: a) Is activation of IKK kinase complex up-regulated in the phorbol ester treated samples? b) Is IkB degradation enhanced? c) Are there post-translational modifications on the NF-kB molecule that can enhance its DNA binding activity.

2) To characterize physiological outcome that NF-kB super-induction will result in. We will test the expression of NF-kB target genes in the presence or absence of phorbol ester treatment. We will also determine the chemo- and radiosensitivity of human cancer cells under condition in which super induction is operational.

Understanding this novel aspect of NF-kB signaling will help develop therapeutic approaches for disorders involving in unregulated NF-kB activity.

Akhil Bhalla
Chapman Lab
Pharmacology 901 Student Seminar
Monday, November 11, 2002
12:00 Noon
Genetics Building - 1111 Auditorium
425 Henry Mall

Analysis of the divalent cation sensors that regulate exocytosis

Abstract: Ca2+ triggers the release of neurotransmitters and hormones from neurons and neuroendocrine cells. The sequence of events linking the influx of Ca2+ to exocytosis is still far from being fully understood. Members of the synaptotagmin (syt) gene family have been proposed to function as the Ca2+-sensors that regulate release. To date, thirteen isoforms of syt have been identified.

Classical electrophysological studies indicated that Sr2+ and Ba2+ are able to trigger exocytosis from neurons. For example, Sr2+ drives slow asynchronous release from some neurons; it was hypothesized that this response was mediated via the Sr2+ sensing ability of syt III. This raised the idea that there might be different sensor for rapid and slow transmission. However, differences in the way that Sr2+ triggers release could be due to differences in the rate of influx through channels, differences in the buffer capacity in the cytosol, or different rates of sequestration/extrusion. Also, the ability of distinct syt isoforms to sense Ca2+ vs. Ba2+ and Sr2+ remains an open issue; there are no clear data to indicate whether any syts can sense metals other than Ca2+. Here, we address this problem by assaying the ability of different cations to activate secretion from cracked PC12 cells. This system overcomes the limitations outlined above. We have also determined which syt isoforms are expressed in these cells and have assayed the metal selectivity of these isoforms. Our preliminary data indicate that syts I and IX act as Sr2+ sensors. Having fully characterized this system, our long term goal is to determine whether changing the divalent cation selectivity of the syt compliment in PC12 cells results in a concomitant change in the metal selectivity of the fusion reaction. These experiments will conclusively answer the question: is syt the metal sensor for exocytosis? This approach will also be used to try to dissect apart other Ca2+-triggered reactions that regulate cell surface membrane traffic (e.g. the metal sensor for adaptation)

Hogune Im
Bresnick Lab
Pharmacology 901 Student Seminar
Monday, November 25, 2002
12:00 Noon
Genetics Building - 1111 Auditorium
425 Henry Mall

Establishment of a Broad Hypoacetylated Chromatin Domain

Organization of DNA into chromatin has important implications for establishing and maintaining cell- and tissue-specific patterns of gene expression. We are using three tissue-specific loci (b-globin, GATA-2, and Notch-4) as models to study transcriptional regulation in the context of native chromosomal domains. The ß-globin locus control region (LCR), which consists of set of DNaseI hypersensitive sites with many transcription factor binding sites, is critical for high-level expression of the globin genes. We hypothesized that the LCR recruits diverse chromatin remodeling enzymes, which mediate long-range transactivation by establishing a broad histone modification pattern. To elucidate the role of histone acetylation/deacetylation in LCR function, chromatin immunoprecipitation (ChIP) analysis was used to measure the acetylation state of the endogenous murine b-globin locus in 14.5 dpc fetal liver and in mouse erythroleukemia (MEL) cells, two adult erythroid cell systems. The adult genes bmajor and bminor and the upstream LCR resided in hyperacetylated chromatin, while the central region of the locus containing the embryonic Ey and bH1 genes was hypoacetylated. Histone deacetylase (HDAC) inhibitor experiments suggested that HDACs are responsible for maintaining low level H4, but not H3, acetylation in the central subdomain. Since little is known about the establishment of broad histone modification patterns, we asked whether the differential regulation of H3 and H4 acetylation applies only to the promoter or to the entire subdomain. The HDAC inhibitor trichostatin A induced H4, but not H3, hyperacetylation at multiple sites within the subdomain in erythroid cells. The hematopoietic factors p45/NF-E2, GATA-1, and EKLF, which function through cis-elements of the b globin locus, were not required for induction of H4 hyperacetylation. However, H4 hyperacetylation was not induced in a non-erythroid cell line. Analysis of chromatin structure within the subdomain revealed low accessibility to restriction endonucleases and nearly complete CpG dinucleotide methylation. Induction of H4 hyperacetylation did not restore hallmark features of transcriptionally active chromatin. We propose that an HDAC-dependent surveillance mechanism counteracts constitutive HAT access, thereby maintaining H4 hypoacetylation throughout the subdomain. However, H4 hypoacetylation does not suffice to establish the inaccessible chromatin structure of the subdomain. To further test our model, we will examine the structure of the globin locus in homogenous populations of embryonic erythroid cells generated via in vitro differentiation of mouse embryonic stem cells. This system will allow one to implement targeted deletions of sequences hypothesized to be critical for establishing and maintaining functionally relevant features of chromatin architecture within the domain.

References:

• 1. Forsberg, E.C., et al., Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain. Proc Natl Acad Sci U S A, 2000. 97(26): p. 14494-9.
  
• 2. Im, H., et al., Histone Deacetylase-Dependent Establishment and Maintenance of a Broad Low-Level Histone Acetylation Within a Tissue-Specific Domain. Biochemistry, 2002. (in press)
  
• 3. Kennedy, M., et al., A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature, 1997. 386(6624): p. 488-93.

Katarzyna Modzelewska
Keely Lab
Pharmacology 901 Student Seminar
Monday, December 2, 2002
12:00 Noon
Genetics Building - 1111 Auditorium
425 Henry Mall

Ack-1 signaling downstream of the a2b1 integrin

It has been established that phosphorylation of p130Cas is a key event in integrin signaling leading to cell migration. We have found that the Cdc42 effector - Ack-1 (Cdc42 associated kinase) plays a role in collagen-stimulated phosphorylation of p130Cas, since kinase dead Ack-1 blocked p130Cas phosphorylation. To determine the role of Ack-1 in integrin signaling, we looked at Ack-1 phosphorylation and its association with other signaling molecules downstream of integrin stimulation. Ack-1 phosphorylation was increased upon collagen stimulation. Surprisingly, this event was not regulated by the Cdc42 GTPase. We found, however, that Ack-1 phosphorylation was regulated by both Src and FAK kinases. Src kinase inhibitor PP2 decreased Ack-1 phosphorylation. Ack-1 phosphorylation was also diminished in the SYF cells (Src, Yes, Fyn -/-) as compared to SYF cells over-expressing c-Src. Phosphorylation of Ack-1 was minimal in the FAK -/- cells, which suggests a role for the kinase in Ack-1 activation. To further investigate the role of Ack-1 in integrin signaling we looked at its association with focal adhesion kinase (FAK), Crk associated substrate (p130Cas) and Src kinase. Ack-1 could be co-immunoprecipitated with both FAK and Src, as well as with p130Cas. FAK and p130Cas association with Ack-1 is minimal in the absence of collagen and increases significantly upon a2b1 integrin stimulation. This is in contrast to the association of Ack-1 with Src, which seems not to be regulated by collagen. Interestingly, Ack-1 association with Src, FAK and p130Cas appears to be independent of Cdc42 activation state. Furthermore, we find that the interactions between Ack-1 and p130Cas and Ack-1 and FAK do not require Src, since they can be co-immunoprecipitated in the SYF cells. Also, FAK is not necessary for the association between Ack-1 and p130Cas or Src. Our results suggest that Src and FAK regulate Ack-1 phosphorylation but not Ack-1 interactions with its signaling partners, and that Ack-1 contributes to the regulation p130Cas phosphorylation downstream of the a2b1 integrin. We are now in the process of dissecting the binding specificities of Ack-1 SH3 domain as well as its proline rich regions

Erik Peden
Barr Lab
Pharmacology 901 Student Seminar
Monday, January 13, 2003
12:00 Noon
Genetics Building - 1111 Auditorium
425 Henry Mall

Male Sex Behavior in Caenorhabditis elegans: A Model System to Study the Neurobiology and Genetics of Complex Behaviors

Genetic analysis of the sexual behavior of male C. elegans represents a powerful approach to identify genes and ultimately molecules that determine how the nervous system generates and controls complex behaviors (Emmons, 2003). Presently, we are mapping a recessive mutation in C. elegans, designated the sy511 allele, which, in males, interferes with two sensorimotor programs required for successful mating: Response (i.e. mate recognition) and location of vulva (Lov) (Liu and Sternberg, 1995). The sy511 mutant line was originally isolated in an EMS mutagenesis screen designed to uncover genes required for male mating behavior and neuro-sensory integration. Interestingly, only sy511 males exhibit behavioral defects, suggesting that the wild type allele functions exclusively in male specific neurons to regulate sexual behavior. GFP expression experiments rule out a role for sy511 in development, though, as sy511 chemosensory neurons are morphologically normal.

Linkage analysis places sy511 on chromosome III, and genetic (3 factor cross) and physical (SNP) mapping place sy511 within a 2cM interval between -2.2 and -4.2 on the physical map. This interval is spanned by over 20 cosmids and includes greater than 80 genes. To narrow the interval further, sequencing efforts are ongoing to construct a denser SNP map of this region. Our mapping approaches and data will be presented, as will be efforts to further investigate the behavioral phenotypes of sy511 males.

Ultimately, the mapping, cloning and molecular analysis of sy511 is expected to shed light on the neural, genetic and molecular hierarchies of behavior. Future studies will focus on the cellular signal transduction pathways of the gene mutated in the sy511 allele through genetic interactions.

References:

1. Emmons, S.W. and Lipton, J. (2003). Genetic Basis of Male Sexual Behavior. J Neurobiol 54: 93-110.

2. Liu, K.S. and Sternberg PW. (1995). Sensory regulation of male mating behavior in Caenorhabditis elegans. Neuron, Jan 14 (1): 79-89.

Pharmacology 901 Student Seminar
Paul Nuzzi
Huttenlocher Lab
Monday, January 27, 2003
12:00 Noon
140 Bardeen

The Role of Calpain in Neutrophil Migration

Neutrophil migration and chemotaxis are critical steps involved in innate immunity and the development of inflammation. We have examined the role that the calcium-dependent cysteine protease, calpain plays during neutrophil migration. In the absence of exogenous stimulators neutrophils maintain high levels of calpain activity, display a spherical morphology and fail to adhere or migrate on extracellular matrix components such as fibrinogen Fbg). Our results demonstrate that in their resting state, neutrophils display high levels of active calpain, with µ-calpain as the predominent isoform. Using both exogenous chemical inhibitors, as well as an HIV-TAT fusion protein construct, we have been able to modulate calpain activity in primary neutrophils. Contrary to previously examined cell types, calpain inhibition leads to enhanced random migration in the absence of exogenous activators, while disrupting neutrophil chemotaxis. Experiments performed using both pertussis toxin and LY294002, suggest that calpain may function downstream of GPCRs and the PI3K pathway. Interestinly, calpain inhibition also leads to an increase in the cellular levels of active Cdc42 and Rac, suggesting a possible mechanism of action. These data suggest, calpain is important in neutrophil migration and function in neutrophils as a negative regulator. We propose, that constitutively high calpain activity in resting neutrophils normally suppresses cell protrusion and migration. Calpain may function downstream of GPCR and PI3K to regulate migration by inhibiting Cdc42 and Rac. Together, these data reveal a novel function for calpain and suggest that neutrophils can regulate polarization and protrusion during chemotactic migration by spatially regulating calpain activity.

Pharmacology 901 Student Seminar
Bernice Lin
Bradfield Lab
Monday, February 3, 2003
12:00 Noon
140 Bardeen

Understanding the AHR Pathway

The aryl hydrocarbon receptor (AHR) is a prototype member of the PAS (Per-Arnt-Sim) superfamily of proteins. In addition to the signature PAS domain of 200 amino acids, members of this superfamily commonly have a bHLH domain that recognizes and binds specific DNA sequences in enhancer elements. The Aryl Hydrocarbon Receptor (AHR) is known for its ability to mediate toxic effects of industrial chemical byproducts such as halogenated and polycyclic aromatics (i.e. Dioxin). The outcomes of exposure to AHR agonists include, induction of xenobiotic metabolizing enzymes, teratogenicity, immune suppression, tumor promotion, and death. Despite the recognized involvement of AHR in such effects, the causal molecular pathway to toxicity still remains unclear. What also remains unresolved is the true physiological role of the AHR. However, recent work on the AHR-/- does suggest importance in hepatic vasculogenesis.

In the unliganded state, the AHR exists in the cytoplasm, bound to a dimer of hsp90, and Ara9. Previous work has shown hsp90's involvement with AHR to be important in maintaining the correct conformational state for ligand binding. However, the role of Ara9 in the AHR pathway is still unclear. It may be interacting to stabilize AHR, or function as part of a DNA binding complex; important in recruitment of cofactors, or may be acting to mask the nuclear localization sequence (NLS) of the AHR; thereby inhibiting nuclear transport. Developmentally, Ara9 is expressed prior to, as well as in tissues distinct from AHR expression. Therefore, Ara9 may play a role outside of the AHR pathway.

One ongoing project is the creation of a conditional Ara9 knockout mouse. We hope that a knockout will shed light on the importance of Ara9 in the AHR pathway, as well as provide insight into other roles for the Ara9 protein in development.

Previous work in our lab gives evidence for a masked nuclear localization signal that can be unmasked by insertions within the a-helix at the basic region of the bHLH domain of AHR. As previously stated, Ara9 may be responsible for masking of this nuclear localization signal. We have constructed several DNA-binding mutants of the AHR by inserting alanine's at this bHLH site to rotate the helix and uncover a masking/unmasking phenomenon.

1. Carver, L.A., LaPres, J.J., Jain, S., Dunham, E.E., and Bradfield, C.A. (1998) J. Biol. Chem. 273:33580-87.

2. Gu, Y-Z, Hogenesch, J.B., and Bradfield, C.A. (2000) Annu. Rev. Pharmacol. Toxicol. 40:519-61.

3. Petrulis, J.R., Hord, N.G., and Perdew, G.H. (2000) J. Biol. Chem. 275:37448-53.

Pharmacology 901 Student Seminar
Brandon Burbach
Rapraeger Lab
Monday, February 10, 2003
12:00 Noon
140 Bardeen

Syndecan-1 extracellular domain regulates spreading and migration of breast carcinoma cells

Syndecan-1 belongs to a family of four cell surface heparan sulfate proteoglycans (HSPG) and is thought to regulate epithelial cell adhesion and migration (1). Analysis of HSPG expression in primary human breast carcinomas and cultured breast carcinoma cell lines demonstrates that these cells frequently express or overexpress syndecan-1 (2). Specific molecular function(s) of syndecan-1 within the mammary epithelium, however, remain largely unknown.

The syndecan extracellular domain core protein is highly divergent between family members, and we hypothesized that this domain interacts within signaling complexes that regulate cellular adhesion and migration in a syndecan-type specific manner. To characterize this putative activity, cell adhesion, morphology, and migration was monitored following overexpression of syndecan-1 in T47D, MCF7, and Hs578t human breast carcinoma cells. Syndecan-1, but not syndecan-4, overexpression results in incomplete spreading of cells plated in serum culture, resulting in a rounded shape compared to the well-spread morphology of control cells. Mutational analysis using a variety of syndecan-1 expression constructs in T47D cells traces this activity to a 146 amino acid central domain within the syndecan-1 ectodomain core protein.

Turning to defined matrix ligands for characterization of the molecular mechanism responsible for disrupted spreading, expression of a lipid-anchored syndecan-1 ectodomain construct (GPI-mS1ED) also reduces spreading on and migration across fibronectin and type I collagen, compared with control cells. Preliminary evidence demonstrates that incubation of T47D cells with soluble bacterially expressed syndecan-1 ectodomain fusion protein mimics the effect of syndecan-1 overexpression. This suggests that syndecan-1 interaction with a cell surface signaling partner triggers inactivation of the cell spreading mechanism. To explain the syndecan-1 ectodomain effect, one possibility is that syndecan-1 may block the integrin(s) that promote spreading and migration on these matrix ligands. Surprisingly, several lines of evidence including adhesion and FAK activation assays suggest that integrin activation on the GPI-mS1ED cells does not differ from controls.

These data support a model in which the syndecan-1 extracellular domain core protein interacts with an unidentified cell surface co-receptor, which blocks a downstream switch in the integrin-mediated signaling cascade, in turn disrupting spreading and migration of breast carcinoma cells. Future experiments are aimed at (1) further defining the novel syndecan-1 core protein signaling domain (2) characterizing the molecular mechanism by which spreading is blocked downstream of integrin activation and (3) identification of the putative cell surface syndecan-1 co-receptor.

References:

1. Carey DJ. 1997. Syndecans: multifunctional cell-surface co-receptors. Biochem J. 327(1): 1-16. (Available online http://www.biochemj.org/bj/327/0001/3270001.pdf)

2. Burbach et al. 2002. Syndecan-1 accumulates in lysosomes of poorly differentiated breast carcinoma cells. Matrix Biology. In press.

Pharmacology 901 Student Seminar
Yukiko Muroi
Walker Lab
Monday, February 17, 2003
12:00 Noon
140 Bardeen

Investigation of Conformational Changes in Ligand-Gated Ion Channels

The goal of my research project is to investigate the conformational changes of one of the ligand-gated ion channels, the GABAA receptor. Approaches we are using are a site-specific incorporation/attachment of a fluorophore into the GABAA receptor. These fluorescent-tagged channels will be activated by application of ligands. The movement of the probe within the channels will then be observed by measuring the fluorescence intensity. The intensity is expected to change according to the probe's environment. The probe we are using should increase its fluorescence when it is transferred from a polor to a nonpolar medium. Moreover, simultaneous measurement of the current will be used to relate the movement of the probe to the receptor conformational state.

The first approach is a site-specific incorporation of a fluorescent unnatural amino acid into the channels. This technique requires chemical acylation of nonsense suppressor tRNA with an unnatural amino acid (7-nitrobenz-2-oxa-1, 3-diazole-4-yl aminoacylated to diaminopropionic acid), and expression of a mutant form of the channel in Xenopus oocytes. Then the amino acid is incorporated into proteins by expressing mRNA harboring an amber nonsense mutation.

Another approach we have taken to reveal the structural change of GABAA receptor is a site-specific attachment of fluorophore to substituted cysteine residues. A cysteine residue is introduced at a specific site in the receptor subunit. Then the residue is fluorescently labeled with a thiol-reactive probe. Again, when the environment of this fluorophore changes upon receptor activation, some changes in the fluorescence intensity should be detected.

Pharmacology 901 Student Seminar
Deanna Beauvais
Rapraeger Lab
Monday, March 3, 2003
12:00 Noon
140 Bardeen

Syndecans are a family of transmembrane cell surface heparan sulfate proteoglycans that have important regulatory roles in cellular differentiation, proliferation, cell-cell and cell-matrix adhesion, cell migration and the regulation of cell morphology. While it is clear that the syndecan heparan sulfate chains are essential for matrix binding, less is known about the signaling role of their core proteins during cell adhesion and reorganization of the actin cytoskeleton. To mimic syndecan-specific cell adhesion, MDA-MB-231 human mammary carcinoma cells were plated on antibodies against syndecan-4 (mAb 150.9) or syndecan-1 (mAb B-B4). While cells adherent via syndecan-4 spread, cells adherent via syndecan-1 do not. However, cells adherent via syndecan-1 can be induced to spread by Mn2+, suggesting syndecan-1 adhesion mediated signaling requires the activation of a b1 or b3 integrin partner. Surprisingly, pre-treatment of cells with function activating b1 antibodies (mAb TS2/16) does not induce syndecan-1 mediated spreading, whereas function blocking b1 integrin antibodies (mAb P5D2 or mAb 13) trigger a spreading response, suggesting that a b1-to-b3 integrin cross-talk may be involved. Indeed, blockade of b1 integrin activation (in specific a2b1 integrins) induces avb3 integrin activation, as detected by soluble fibrinogen binding, and this activity is required for syndecan-1 adhesion mediated signaling as treatment with avb3 blocking antibody, mAb LM609, inhibits cell spreading. Spreading in response to syndecan-1 ligation occurs in the absence of serum and is independent of integrin-mediated adhesion as spreading is unaffected by treatment with EGTA or cycloheximide. However, spreading can be inhibited by competition with soluble mAb B-B4 and with soluble murine syndecan-1 ectodomains. Further, mutational analysis of the syndecan-1 core protein indicates spreading requires a 35 a.a. region within the syndecan-1 ectodomain, yet does not require the proteoglycan’s heparan sulfate glycosaminoglycan (GAG) chains. These data suggest that in response to an organized extracellular matrix, the ectodomain of the syndecan-1 core protein participates in the formation of a signaling complex with avb3 integrins that transduces a signal to reorganize the actin cytoskeleton. Signaling via this complex is negatively regulated by b1 integrins and this signaling mechanism may be a critical regulator of cell migration and invasiveness in development and in cancer.

Pharmacology 901 Student Seminar
Siobhan Wilson
Keely Lab
Monday, March 31, 2003
140 Bardeen
12:00 Noon

R-Ras Regulates Adherens Junction Formation in Breast Epithelial Cells.

In normal breast tissue, epithelial cells polarize into ducts. This process can be modeled in vitro by culturing cells in 3D matrices, under which conditions breast epithelial cells recapitulate the formation of duct-like tubules. Key in tubulogenesis is the creation of cell-cell contacts (including adherens junctions, AJs) and cell-extracellular matrix contacts (including focal adhesions, FAs). AJs and FAs are established in balance in normal tissue to create polarized, differentiated cells. Cellular events that disturb the coordination of AJs vs. FAs may lead to aberrant cell behavior, cell migration, and carcinogenesis.

R-Ras is a small GTPase that regulates integrin function and promotes cell migration. Little is known regarding the role of R-Ras in epithelial polarity or transformation. I showed that R-Ras activation disrupted cell-cell contacts and enhanced cell-matrix contacts in MCF-10A breast epithelial cells. In both 2- and 3-dimensional cultures, the localization of E-cadherin, b-catenin, a-catenin, ZO-1, and other proteins to AJs was altered in cells expressing activated R-Ras. Coincident with this, there was a pronounced increase in FA formation. Of particular interest, we observed the relocalization of several proteins, including the _2_1 integrin and vinculin, from cell-cell to cell-matrix contacts. Endogenous R Ras localized in significant amounts to the apical surface of epithelial cells in normal breast tissue sections. To further understand how R-Ras regulates epithelial phenotype, I am using various approaches to characterize the function of endogenous R-Ras in establishing effects on epithelial polarity, AJs, and FAs.

Given the phenotype of R-Ras overexpression and the localization of R-Ras in vivo, I believe that endogenous R-Ras may play a role in establishing polarity in normal epithelial cells, namely in regulation of the balance between cell-cell contacts and cell-matrix contacts.

Pharmacology 901 Student Seminar
Jaehyung (Gus) Cho
Deane F. Mosher's lab
Monday, April 7, 2003
140 Bardeen
12:00 Noon

Matrix assembly of fibronectin (FN)

Matrix assembly of fibronectin (FN) is associated with diverse processes including inflammation, wound repair, malignant metastasis, microorganism attachment and thrombosis. The N-terminal 70-kDa region of FN (70K) plays an important role in initial binding of FN to cell surfaces and in matrix assembly of FN. The cell surface binding sites for 70K, however, remain to be identified.

Platelet adhesion and aggregation at the site of vascular injury are crucial events, leading to the formation of a hemostatic plug and subsequent arrest of bleeding or thrombotic diseases in serious
conditions. A role of FN for platelet functions has long been suspected. Recently, our group demonstrated that adherent platelets have the capacity to assemble to FN (1). FN played a role in formation of platelet aggregates in arterioles of mice lacking both von Willebrand factor and fibrinogen (2), and plasma FN was shown to promote thrombus growth and stability in injured arterioles (3).

To identify platelet proteins that bind to the 70K and may participate in matrix assembly, we investigated ligand blotting of FITC-labeled 70K to platelet fractions previously separated by gel filtration in SDS. Specificity was assessed by inclusion of unlabeled 70K or FUD, a recombinant 49-residue peptide that binds to 70K and blocks its interaction with cells. 70K specifically bound to a limited number of bands. In 2D electrophoresis, similar to what was seen in ligand blotting, FITC labeled 70K bound to several proteins (40-, 65-, 80-, and 140-kDa) in platelet fractions, and the binding was partially or completely inhibited by FUD. The 140-kDa region contained intracellular molecules such as filaminA and talin as well as membrane proteins such as glycoprotein Iba when arrayed by mass spectrometry. The 65- and 80-kDa proteins were identified as fibrinogen Bb and Aa polypeptides, respectively. Whether these candidate proteins specifically bind to 70K and account for the association of 70K to cell surfaces will be clarified by further studies.

References:
1. Olorundare O.E. et al. Blood 2001;98:117-24

2. Ni H. et al. J Clin Invest 2000;106:385-92

3. Ni H et al. PNAS 2003;100:2415-9

Pharmacology 901 Student Seminar
Reema Jasuja
Greenspan Lab
Monday, April 7, 2003
140, Bardeen
12:00 Noon

Prodomains of BMP-1/mTolloid like Proteases in Extracellular Matrix Deposition and Growth Factor Signaling

When bone morphogenetic activity in bone extracts was highly purified, it was found to correspond to peptides whose sequences lead to cloning of cDNA's for the Bone Morphogenetic Proteins (BMP's). BMP-1 was found to be an extracellular zinc dependent metalloprotease while all other BMP's (which now number over 20) are TGFb-like molecules. BMP-1 has been found to biosynthetically processes precursors into the mature functional forms of various proteins important to formation of the extracellular matrix. Such proteins include the major and minor fibrillar collagens, laminin 5, lysyl oxidase, and biglycan. BMP-1 is synthesized with an N-terminal prodomain that is removed by Furin-like proprotein convertases, presumably activating the protease. Interestingly, all characterized peptide sequences for the form of BMP-1 that co-purifies with TGFb-like BMP's in osteogenic fractions have been from the prodomain region. To gain more insight into possible functional roles for the BMP-1 prodomain in vertebrates, we have evaluated the proteolytic activity and the binding properties of a furin site-mutated form of ProBMP-1 (ProBMP-1SQQ), that retains its prodomain sequence. We found that ProBMP-1SQQ is efficiently expressed and secreted but is proteolytically inactive. Moreover, ProBMP-1SQQ was found to act as a dominant negative version of BMP-1, suggesting tight binding of substrates in the absence of proteolytic activity. Recently, we have shown that ProBMP-1SQQ binds to TGFb-like BMPs as well.

Pharmacology 901 Student Seminar
Haichuan Duan
Jefcoate Lab
Monday, April 14, 2003
140 Bardeen
12:00 Noon

Post-transcriptional regulation of the Steroidogenic Acute Regulatory Protein (StAR) gene

Steroidogenesis is the process by which specialized cells in steroidogenic tissues (such as adrenal cortex, gonads and placenta) generate steroid hormones from cholesterol. The rate-limiting step in steroidogenesis is transfer of cholesterol from the outer mitochondrial membrane to inner mitochondrial membrane where the first enzyme for steroid hormone biosynthesis, P450scc, is located. The Steroidogenic Acute Regulatory protein (StAR) is a crucial mediator of cholesterol transfer through the aqueous intramitochondrial membrane space. In rodent adrenocortical and testis Leydig cells, two major transcripts of StAR, 3.5kb and 1.6kb, are expressed. These two transcripts arise from alternative polyadenylation and differ only in the length of the 3' untranslated region (UTR). An AU-rich element (AURE), which has been shown to regulate the stability of many cytokine and proto-oncogene mRNAs, is located near the end of the long 3'UTR of 3.5kb transcript but not the 1.6kb transcript. In luciferase/StAR 3'UTR chimeric constructs, the long 3'UTR of StAR but not the short one was found to destabilize luciferase message 2 to 3 fold. Deletion of AURE from long 3'UTR, however, did not abolish its destabilizing effect, suggesting other elements in StAR long 3'UTR are responsible for its instability under basal conditions. Prolonged 8Br-cAMP stimulation of mouse adrenocortical Y-1 cells and Leydig tumor MA-10 cells leads to selective decline of 3.5kb StAR transcript, which we hypothesize is mediated through AURE. A candidate trans-acting protein factor for the late destabilization on 3.5kb StAR transcript is TIS11b, a member of the CCCH double zinc finger family of proteins that have been shown to mediate stability regulation of many cytokine mRNAs through binding to their AUREs. TIS11b is found to be rapidly induced by 8Br-cAMP in Y-1 cells, preceding the late decline of 3.5kb StAR transcript. Cotransfection of TIS11b with luciferase/StAR 3'UTR chimeras show TIS11b can destabilize luciferase chimera with full-length long StAR 3'UTR but not AURE deleted 3'UTR. In another study, several MAPK pathway kinases were shown to selectively increase the level of 3.5kb StAR mRNA. Whether this involves AURE remains to be determined. Both forms of StAR mRNA are stabilized upon treatment with various transcriptional inhibitors, which has prevented direct measurements of StAR message degradation kinetics. Alternative approaches for the assay will be discussed.

Pharmacology 901 Student Seminar
Jing Wu
Bresnick Lab
Monday, April 14, 2003
140 Bardeen
12:00 Noon

Endothelial-Specific Regulation of Notch4 Transcription

Notch signaling plays important roles in cell fate determination. Notch receptor family members, Notch1-4, share a conserved domain structure and have some overlapping functions, but can also function distinctly. In situ hybridization experiments suggested that Notch4 is expressed specifically in endothelium. However, very little is known about mechanisms that confer endothelial-specific transcription. We are interested in how Notch4 transcription is regulated specifically in endothelial cells, especially the role of chromatin remodeling in controlling transcription. The specific histone modification pattern of the human Notch4 locus in human umbilical vein endothelial cells (HUVECs) was identified and compared to that in HeLa cells, a non-endothelial cell line. The strong enrichment of histone modification in HUVECs was restricted to the promoter and to the first three exons. Based on the histone modification pattern and sequence conservation, we hypothesize that three regions are important in conferring endothelial-specific transcription: an upstream conserved region, the promoter, and the first intron. Reporter assays were used to measure the functions of these regions. In transient transfection analysis, the Notch4 promoter conferred preferential activity in HUVEC compared to HeLa. Furthermore, in vivo transgenic analysis in 10.5 dpc mouse embryos suggested that the Notch4 promoter alone is inactive. However, a construct containing the Notch4 promoter and the conserved region yielded a typical vascular staining pattern. These results suggest the following model. The Notch4 promoter can confer endothelial-specific activity, but its activity is repressed by higher-order chromatin structure in non-endothelial cells. A regulatory element, such as a locus control region in the upstream region, is responsible for the endothelial-specific transcription regulation in vivo. Chromatin remodeling enzymes recruited to this region in endothelial cells acetylate or methylate the promoter, which counteract the repression in the promoter and thus stimulate transcriptional activation. We plan to identify the regulatory elements and specific transcription factors that establish the endothelial-specific chromatin structure and transcription, which will provide basic insights into tissue-specific transcriptional mechanisms and will allow for the construction of vectors for expressing genes in the vasculature.

Pharmacology 901 Student Seminar
Melissa Martowicz
Bresnick Lab
Monday, April 21, 2003
140 Bardeen
12:00 Noon

Notch signaling occurs through binding of the transmembrane Notch receptor to a ligand (Jagged or Delta) on an adjacent cell.

Following binding, the intracellular domain of Notch (NIC) is liberated by proteolytic cleavage. NIC then translocates to the nucleus where it binds the transcriptional repressor CSL, which leads to transcription of Notch target genes. Notch signaling is evolutionarily conserved and controls developmental processes such as vasculogenesis, hematopoiesis, and myogenesis. In addition, activated forms of Notch resulting from expression of the intracellular domain are implicated in oncogenesis. Our laboratory is interested in identifying novel modes of crosstalk between the Notch pathway and other cell signaling mechanisms that control hematopoiesis and vasculogenesis. Recently, it was found that the intracellular domain of Notch-1 (NIC-1) represses interleukin-8 transcription in human K562 erythroleukemia cells (1). Further analysis revealed that this repression resulted from an action of NIC to antagonize the Activator Protein-1 (AP-1) transcription factor (2). AP-1 is a critical regulator of multiple genes involved in immune and inflammatory reactions. Ha Ras-induced activation of AP-1 in human cell lines was repressed by NIC-1, and repression required a domain of NIC-1 known only to mediate transcriptional activation. Based on these results, and genetic evidence in Drosophila suggesting opposing actions of Notch and Ras, we hypothesized that there is bi-directional crosstalk, in which Notch represses Ras and components of the Ras/Raf/MEK pathway repress Notch (3). We asked whether components of the Ras pathway block Notch mediated transcriptional activation. A constitutively active MEK-1 (MEK-1-R4F) strongly repressed NIC activity in K562 cells. In contrast, MEK-1-R4F did not inhibit basal transcription nor transcriptional activation mediated by a different activator. Ha-Ras and Raf did not repress NIC mediated transactivation, suggesting that repression of NIC by MEK is independent of these kinases. Western blot analysis shows that expressed NIC levels are decreased by co-expression of MEK-1 R4F. RT-PCR measuring expressed NIC mRNA levels indicates that co-expression of MEK-1-R4F does not impact on the expression of NIC. These results support the hypothesis that there is bi directional crosstalk between between Notch and Ras/Raf/MEK signaling, and that NIC levels are regulated by constitutively active MEK-1. Future studies will define the mechanism and physiological implications of this new mode of Notch regulation.

References:
1. Lam, L., Ronchini, C., Norton, J., Capobianco, A., and Bresnick, E. (2000) J. Biol. Chem 275, 19676-19684.

2. Chu, J., Jeffries, S., Norton, J., Capobianco, A., and Bresnick, E. (2002) J. Biol. Chem. 277, 7587-7597.

3. Zecchini, V., Brennan, K., and Martinez-Arias, A. (1999) Curr. Biol. 9, 460-469.

Pharmacology 901 Student Seminar
Jill Humston
Ervasti Lab
Monday, April 21, 2003
140 Bardeen
12:00 Noon

Functional improvement of dystrophic muscle by myostatin blockade

By pharmacologically manipulating the signaling events important for regulating muscle differentiation, growth and development, a recent paper in Nature by Bogdanovich S et al, "Functional improvement of dystrophic muscle by myostatin blockade," hopes to provide a novel therapeutic approach for Duchene Muscular Dystrophy, an X-linked recessive progressive muscle wasting disorder.

Pharmacology 901 Student Seminar
Angela Mabb
Miyamoto Lab
Monday, May 5, 2003
140 Bardeen
12:00 Noon

A Potential Role for a Ubiquitin Conjugating Enzyme 2 (Ubc2) in NF-kB Activation Pathways

NF-kB is a transcription factor that regulates various genes such as those that are involved in growth control, immune function, and apoptosis.  NF-kB is activated by a wide variety of stimuli, such as TNF-a, IL-1, LPS, and viruses.  NF-kB has also been shown to be activated through DNA damaging agents that induce double strand breaks, such as IR, camptothecin, and etoposide (VP16).  These activators signal from the nucleus to activate a complex, known as the IkappaB kinases that are necessary for NF-kB activation.  Ubiquitins have been shown to be important modulators of the NF-kB pathway, however, more studies need to be done to further characterize the role of ubiquitination in the NF-kB pathway.  Recently, our lab has found that two ubiquitin conjugation enzymes (E2's), Ubc2 (Rad6) and Ubc13, may play a role in the NF-kB pathway upon addition of DNA damaging agents, camptothecin and VP16.  Both of these E2's have been shown to tether multiple ubiquitin chains to a protein substrate via K63.  This modification does not target a protein substrate for degradation, but initiates specific signaling events that have been shown to be responsible for DNA repair, endocytosis, and subsequently NF-kB activation.  Further analysis needs to be performed to determine the role of Ubc2 (Rad6) and 13 in DNA damage induced NF-kB activation.

References:
1. Karin, M., Ben-Neriah, Y.  (2000)  Ann. Rev. Immunology.  18, 621-663.

2. Pickart, C.  (2001)  Annu. Rev. Biochem.  70, 503-533.

3. Broomfield, S., Hryciw, T., Xiao, W.  (2001)  Mutation Research.  486, 167-184.--

Pharmacology 901 Student Seminar
Shelby O'Connor
Miyamoto Lab
Monday, May 19, 2003
140 Bardeen
12:00 Noon

Mechanism of Constitutive NF-kB (p50/c-Rel) Activation in B cell Development

Constitutive NF-kB activity is crucial for B cell development. However, the mechanism for this activity has been a mystery since the original discovery of NF-kB in this B cell system. We have previously found the maintenance of constitutive NF-kB (a p50/c-Rel heterodimer) activity depends on rapid, continuous, and selective degradation of IkBa in both normal and transformed B cells. Most known inducible pathways require the N-terminal signal response domain for IkB kinase (IKK)-dependent phosphorylation. Dually phosphorylated IkBa is recognized by the E3 ubiquitin ligase b-TrCP. Poly-ubiquitination of IkBa on its N-terminal lysine(s) by the b-TrCP complex results in subsequent proteolysis by the 26S proteasome. Constitutive IkBa degradation in B cells also appears to require IKK activity and its phosphorylation sites on IkBa. Surprisingly, however, this degradation in B cells is independent of proteasome activity. Furthermore, the N-terminal lysines that are essential for inducible degradation are dispensable for constitutive IkBa de