2002 Signal Transduction Research Training Symposium

October 7, 2002 : Fluno Center, Madison, WI

Sponsors: Molecular and Cellular Pharmacology Training Program, the UW Comprehensive Cancer Center, the Department of Pharmacology, the Department of Physiology, Biosource International, the UW Cardiovascular Research Center, and the Promega Corporation

Symposium Committee: Patricia J. Keely (Chair), Richard Burgess, Alan Rapraeger, David Wassarman, Sujatha Venkataraman, Lynn Squire.

Symposium Keynote

“Transcriptional Control of Cardiac Development and Disease”

Dr. Eric N. Olson
University of Texas Southwestern

Student Symposium Talks

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

Jianlin Chu1, Shawn Jeffries2, Jason E. Norton1, Anthony J. Capobianco2, and Emery H. Bresnick

1University of Wisconsin Medical School, Department of Pharmacology, Molecular and Cellular Pharmacology Program, Madison, Wisconsin 53706 and 2University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry and Microbiology, Cincinnati, Ohio 45267-0524

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.

Wnt signaling and syndecan-1 affect the number of mammary progenitor cells, with important implications for mammary tumor development.

B. Liu, S.S. Khwaja, V. Leatherberry, C.M. Alexander
Department of Oncology for Cancer Research,
University of Wisconsin-Madsion, Madison, WI 53706

Dysregulation of the Wnt signaling pathway resulting in activation of b-catenin has been found in numerous human cancers including breast, colon, uterus, skin, prostate, and medulloblastoma. Overexpression of Wnt-1 or nondegradable b-catenin in the mammary gland of a virgin mouse induces a hypermorphic phenotype similar to that of a mid-pregnant gland and a subsequent development of mammary adenocarcinomas. Since Wnt signaling has been implicated in stem cell development in the intestine and the skin, we propose that the hypermorphic Wnt-1 and b-catenin transgenic glands may be caused by an aberrant regulation of mammary progenitor cells. Here, we show that mammary gland progenitor cells as measured using Hoechst-33342 dye exclusion (“side-population” (SP)) are increased in Wnt-1 and nondegradable D89b-catenin transgenic hyperplastic glands and tumors. This SP population is also increased in response to soluble Wnt in cultured primary mammary epithelial cells (MECs). The SP population is increased by 40% in Wnt-3A conditioned media relative to that of the control media. Interestingly, the number of SP cells is reduced by 50% in cultured MECs isolated from mammary glands of mice that have a targeted null mutation for the heparan sulfate proteoglycan, syndecan-1 (Sdc1). Moreover, the number of mammary tumors induced by D89b-catenin are reduced in the Sdc1 -/- background relative to that of the Sdc1+/+ background. The tumor latency is significantly delayed in the Sdc1 -/- mice relative to that of the Sdc1 +/+ mice. These data suggest that Wnt signaling and Sdc1 affect the number of mammary progenitor cells that may have an important effect on mammary gland tumorigenesis.
Bob Liu w/Caroline Alexander

Role of Jumonji in Murine Cardiac Development

Jonathan C. Kraus, Jason M. Keaton, Gary E. Lyons, and Youngsook Lee
Department of Anatomy, University of Wisconsin-Madison

Molecular analyses demonstrate that the jumonji (jmj) gene product functions as a transcriptional regulator that contains a transcriptional repressor domain and is a localized to the nucleus. CASTing and gel retardation assays identified high and low affinity JMJ DNA binding motifs, some of which are found in cardiac expressed genes.

To investigate the role of jmj during development, we characterized mice homozygous for the jmj mutation by insertion of the gene trap vector. The jmj homozygous mouse embryo with a heterogeneous genetic background showed heart malformations including ventricular septal defect, noncompaction of the ventricular wall, double outlet right ventricle, and dilated atria. The homozygous jmj mutants died soon after birth apparently due to respiratory insufficiency, which may result from rib and sternum defects in addition to the heart defects. In situ hybridization and lacZ staining in embryos showed expression of jmj in the myocardium that correlated well with location of defects observed in the hearts of homozygous mice. The homozygous mutants failed to express the jmj transcript in all organs and tissues except in the nervous system, resulting in a tissue-specific disruption of jmj.

To examine the effect of genetic background on the mutant phenotype, mice with the jmj mutation were bred onto a homogenous Black C6 background. These mutant embryos developed severe, whole body edema by embryonic day (E) 13.5 and all died by E 14.5. These mutant mice exhibited similar phenotypic cardiac abnormalities including ventricular septal defect and double outlet right ventricle. These mutant embryos also appeared to be anemic. Other phenotypic abnormalities included small liver and a decreased body size.

These data demonstrate that jmj is essential for normal heart development and function and, therefore, jmj mutant mice may serve as an animal model for human birth defects including congenital heart defects.

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?

Tony T. Huang and Shigeki Miyamoto

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.

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

Indra Lim and Johannes Hell
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.

Rho, ROCK, and FAK regulate cellular contractility and focal adhesion formation to promote breast epithelial ductal morphogenesis

Michele A. Wozniak, Radhika Desai, and Patricia J. Keely
Department of Pharmacology, University of Wisconsin, Madison, WI 53706

Breast epithelial cells differentiate into polarized duct-like tubules when cultured in a floating three-dimensional collagen matrix. Remarkably, this differentiation does not occur when the cells are cultured in the same collagen matrix that is attached to the culture dish, suggesting that the cells sense the physical rigidity of their environment and activate specific signaling pathways in response. Breast cell sensitivity to the biophysical properties of the extracellular matrix (ECM) is significant because patients with dense fibrous breast tissue are at increased risk of developing breast carcinoma. Relatively little is known about how cells sense and respond to the rigidity of their surrounding extracellular matrix. Breast epithelial cells cultured in a flexible matrix, which promotes tubulogenesis, contract the collagen matrix and lose FAK phosphorylation at Y397. In contrast, cells cultured in a rigid matrix do not form tubules and retain FAK phosphorylation at Y397 localized to focal contacts. The ROCK inhibitor, Y27632, blocks tubulogenesis and collagen gel contraction and results in increased phosphorylation of FAK at Y397. This data suggests a functional link between ROCK-mediated cellular contractility, loss of FAK phosphorylation at Y397, and tubulogenesis. To determine the mechanism involved in this response, Rho activity was analyzed. Rho activity was high in cells presented with a rigid collagen matrix, whereas cells encountering flexible collagen fibers down regulate Rho activity within 60 minutes. Surprisingly, inhibiting ROCK with Y27632 prevents this decrease in Rho activity. Since ROCK is an effector for Rho, this suggests a regulatory feedback loop in which ROCK-mediated contractility diminishes Rho activity. Our data demonstrate the novel finding that breast epithelial cells sense the rigidity of their environment via ROCK-mediated contractility and a subsequent down regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.

SORCIN, A Modulator Of Excitation Contraction Coupling In Heart

Emily F Farrell, Ana M Gómez1, Anaid Antaramian, Nancy A Benkusky, Lameh Fananapazir2 & Héctor H Valdivia,
University of Wisconsin-Madison, 1INSERM, Montpellier, France, 2NHLBI of NIH, Bethesda, MD.

Sorcin, a 22-kDa Ca2+-binding protein, is expressed in several cell types including cardiomyocytes, but its function is unknown. In vitro, sorcin binds to cardiac RyRs (RyR2) and L-type Ca2+ channels. We thus hypothesized that sorcin may play a role in excitation-contraction coupling in the heart. In saponin-permeabilized mouse cardiomyocytes, sorcin (1 mM) decreased the amplitude and duration of spontaneous Ca2+ sparks, suggesting direct interaction with RyRs in vivo. In voltage-clamped ventricular cells loaded with Fluo-4, sorcin increased the peak and time to inactivation of the L-type Ca2+ current (ICa), decreased cell contraction, and tended to decrease the [Ca2+]i transients. Furthermore, sorcin undergoes Ca2+-dependent translocation from cytosol to membranes, a property that is consistent with sorcin playing a dynamic role in EC coupling. A point mutation in sorcin, F112L, was linked to familial hypertrophic cardiomyopathy (FHC) and hypertension. Unlike wild-type sorcin, which completely inhibited activity of RyRs reconstituted in lipid bilayers, F112L-sorcin was unable to produce the same effect. These results indicate that sorcin plays an important modulatory role in excitation-contraction coupling, a disturbance of which may express itself as overt cardiac malfunction.

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

Blue-leaf Hannah and Deane Mosher
Department of Medicine

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 50ÕC compared to 65ÕC 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.

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

Kun Ling*ã, Renee L. Doughman*ã, Ari J. Firestone*, Matthew W. Bunce*, and Richard A. Anderson*.
*University of Wisconsin-Madison, Department of Pharmacology, Program in Molecular and Cellular Pharmacology, 1300 University Ave. University of Wisconsin Medical School, Madison, Wisconsin, 53706

ã ‘These authors contributed equally to this work’.

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.