2003 Signal Transduction Research Training Symposium

October 7, 2003 : 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, Karl Beyer Trust Fund, and the Promega Corporation.

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

Symposium Keynote

“Cellular Signaling by Tyrosine Phosphorylation”

Dr. Joseph Schlessinger
Professor and Chairman of Pharmacology at Yale University

Student Symposium Talks

Estrogen receptor-a concentration: a non-classical mechanism of receptor activation in breast cancer cells

*Amy M. Fowler, Natalia Solodin, Mara T. Preisler-Mashek, and Elaine T. Alarid
Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA

ERa functions as a ligand-activated transcription factor that mediates estrogen signaling in the breast by controlling the expression of genes involved in growth and differentiation. In the normal breast, expression of ERa protein is restricted to a small proportion of luminal epithelial cells. These ER+ cells express low to intermediate levels of receptor that fluctuate inversely with the concentration of estrogen. In breast cancer, a common phenotype is an expansion of the ER+ population and an inappropriate elevation of ERa protein, the latter of which predisposes patients for poorer prognosis than those with lower levels of receptor. It is currently believed that enhanced ERa expression may provide cells with a proliferative advantage by enabling a greater response to estrogen’s mitogenic signal, thereby promoting their continued survival and expansion. We generated a tetracycline-inducible ERa overexpression system in MCF-7 cells to investigate the consequences of an increase in cellular ERa protein levels on endogenous gene expression and cell proliferation. We found that heightened expression of ERa does not increase the proliferative response to estrogen; yet, in the absence of ligand, it stimulates endogenous gene expression and accelerates growth. The aberrant receptor activity is indeed independent of hormone and AF-2 function since overexpression of a receptor lacking the ligand-binding domain is sufficient. Transactivation by unliganded ERa at high concentrations requires the N-terminal B domain, suggesting an enhancement of the relative contribution of AF-1, yet is independent of serine 104, 106, 118 phosphorylation. The requirement for DNA binding and recruitment of coactivators is currently being investigated. Our studies describe an alternative mechanism of receptor activation independent of ligand binding and cross talk with the mitogen-activated protein kinase pathway. Furthermore, the data suggest that cellular control of ERa concentration is critical for restricting AF-1 receptor activity and preserving ligand-dependent responses.

PIPKIg661 Directly Interacts with the AP2 Complex and Regulates EGF Receptor Endocytosis

*Shawn F. Bairstow*, Daniel Shi, Ari Firestone, Kun Ling, Xiaojing Su and Richard A. Anderson
Department of Pharmacology, University of Wisconsin, *Biomolecular Chemistry Graduate Program, University of Wisconsin.

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. This includes receptor mediated endocytosis at the plasma membrane and sorting of proteins emerging from the trans-Golgi network. The formation of clathrin coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. Once bound to the membrane, these heterotetrameric protein complexes serve as a scaffold for assembly of other proteins responsible for vesicle formation including: membrane curvature, assembly of the clathrin lattice and detachment from the donor membrane. Targeting of the AP complexes to the membrane is also mediated by the presence of the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PIP2). PIP2 is synthesized via the phosphorylation of phosphatidylinositol by phosphatidylinostiol phosphate kinases (PIPK). We have discovered, via yeast two-hybrid screen, a direct interaction between the m2 subunit of the AP2 complex and the PIPK Ig661 isoform. This interaction was confirmed using both the m2 subunit alone and the full AP2 complex via co-immunoprecipitation from HEK293 cells and GST-pull down experiments using recombinant proteins. We also found that tyrosine residues in the 26 amino acid tail of Ig661 are critical for the interaction, specifically Y644 and Y649. As state above, AP2 is responsible for receptor mediated endocytosis at the plasma membrane. We have also observed that expression of catalytically inactive Ig661 in A431 cells inhibits receptor mediated endocytosis of the epidermal growth factor (EGF) receptor. Collectively, this evidence implies an important role for Ig661 in endocytosis of the EGF receptor. The direct interaction between AP2 and Ig661 would lead to concerted targeting of Ig661/AP2 complexes to endocytic sorting motifs. This proposed mechanism would both promote the binding of additional AP2 complexes to the membrane surrounding cargo proteins and enhance the formation of the clathrin coat.

Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a non-additive manner

Ping Wang, Chih-Tien Wang, Jihong Bai, Meyer B. Jackson and Edwin R. Chapman*
Department of Physiology, University of Wisconsin, Madison, WI, 53706

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2-domains, C2A and C2B; each C2-domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2-domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+-requirements for syt•liposome interactions from ~20 to ~40 mM Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect syt•syntaxin interactions, but resulted in a ~50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2- domains of syt cooperate with one-another to trigger release via loop-mediated electrostatic interactions with effector molecules.

L-type Ca2+ channels are Part of a Caveolar Macromolecular Signaling Complex Associated with Caveolin-3 and b2-AR in Ventricular Myocytes

*Ravi C Balijepalli1, Jason D. Foell1, Duane D. Hall2, Johannes W. Hell2 and Timothy J. Kamp1
Department of Medicine, University of Wisconsin1- Madison, Department of Pharmacology, University of Iowa2

Caveolae are specialized structures in the plasma membrane composed of cholesterol, sphigolipids, structural proteins termed caveolins, and associated proteins. Critical molecules for a variety of signaling pathways have been localized to caveolae, but the exact composition of caveolae in cardiac muscle is unknown. Given the importance in cell signaling of intracellular Ca2+, we hypothesized that L-type Ca2+ channels may be localized to caveolae associated with proteins in the cAMP/PKA-signaling pathway. Canine and mouse ventricular tissue or myocytes were employed for biochemical and confocal immunofluorescence studies. Detergent-free sucrose density gradient fractionation of the ventricular tissue or isolated myocytes yielded 12 different 1 ml membrane fractions (5 – 42% sucrose). Western blot analysis of the gradient fractions demonstrated presence of caveolin-3, b2-adrenergic receptor (b2-AR) and Cav1.2 subunits of the L-type Ca2+ channels in fractions 4-6 (15-21% of sucrose). In contrast b1-adrenergic receptors (b2-AR) were more broadly distributed in the gradient. Indirect immuno-fluorescence confocal microscopy of isolated ventricular myocytes showed co-localization of Cav1.2 and caveolin-3 in the surface sarcolemma and most superficial T-tubules. Immunoprecipitation from enriched caveolar membranes using an antibody directed against Cav1.2 was performed to identify associated proteins. Western blot analysis revealed that Caveolin-3 and b2-AR co-immunoprecipitate with Cav1.2 channels, in contrast the b2-AR did not. Alternatively, immunoprecipitation using the caveolin-3 antibody confirmed that Cav1.2 channels and b2-AR co-immunoprecipitate with caveolin-3. Further investigation of the Ca-v1.2 or caveolins-3 immunoprecipitates revealed that adenelyl cyclase (AC), catalytic subunit of protein kinase A (PKA), and protein phosphatase 2A (PP2A) are associated with Cav1.2 and Caveolin-3. Control reactions using nonspecific IgGs did not result in the immunoprecipitation of any of the above proteins. We conclude that Cav1.2 channels are present in caveolae in ventricular myocytes as part of a macromolecular signaling complex containing Caveolin-3, b2-AR, AC, PKA and PP2A.

Crosslinking of Full-Length PDEg Photoprobes to Gat-GTPgS

J. E. Grant*, L. Guo*, M.M. Vestling**, and A.E. Ruoho*.
*Department of Pharmacology, University of Wisconsin-Madison, 53706.
**Department of Chemistry, University of Wisconsin-Madison, 53706.

Dynamic regulation of protein/protein signaling ensures the fidelity and rapidity of vision. In a key step, activated transducin a-subunit (Gat-GTP) must engage the inhibitory g-subunit of the cGMP phosphodiesterase (PDEg), releasing the catalytically active phosphodieseterase a- and b-subunits. As Gat-GTP binds PDEg, recruitment of the Regulator of G-Protein Signaling 9 (RGS9) and the RGS9-specific G-protein b-subunit Gb5 stimulates GTP turnover and Gat deactivation.

Despite numerous technological advances, structural definition of PDEg conformation has remained elusive. The only extant crystal structure incorporating a PDEg fragment (Slep et al., 2001) describes interaction of the PDEg C-terminus during the transition state for GTP hydrolysis (GTP hydrolysis state).

To dissect the interaction of PDEg with Gat in the signaling state, a series of full-length PDEg benzophenones were developed. Full-length PDEg photoprobes were generated semi-synthetically, employing intein-mediated protein ligation. Each photoprobe incorporates one benzophenone substitution at a hydrophobic amino acid, as well as a biotin affinity tag at the extreme C-terminus.

Limited proteolytic digestion of crosslinked species, assessed by avidin/biotin far western detection methods, indicates a novel interaction between PDEg and the Gat-GTPgS N-terminus. Preliminary analysis of crosslinked fragments by a biotin affinity mass spectrometric method support this conclusion.

Sujatha Venkataraman

Ataxia-telangiectasia (A-T) is an inherited syndrome of cancer, genetic instability, immune deficiency and neurodegeneration caused by mutations in ATM. Defects in ATM are associated with defective cell cycle checkpoint regulation and cell survival in response to ionizing radiation (IR) and radiomimetic drugs. Although defective cell cycle checkpoint regulation and associated genomic instability presumptively contribute to cancer susceptibility in A-T, the mechanism of neurodegeneration in A-T is not well understood. Here we describe a direct link between ATM and the neuron survival factor CREB. ATM phosphorylated CREB in vitro on three residues proximal to the kinase-inducible domain. ATM-mediated phosphorylation of CREB in vivo was induced by IR and H2O2 and occurred by a processive mechanism. IR-induced phosphorylation of CREB correlated with 2-fold decrease in CREB trans-activation potential and reduced interaction between CREB and its transcriptional co-activator, CBP. In contrast, a CREB3A mutant containing alanine substitutions at ATM phosphorylation sites displayed enhanced trans-activation potential, resistance to inhibition by IR, and increased binding to CBP. Our findings are consistent with a model whereby ATM-mediated phosphorylation antagonizes CREB function in response to DNA damage or oxidative stress. We have recently found that the ATM-CREB pathway is activated in primary cerebellar granule neurons isolated from mice and our currently investigating the role of this pathway in regulating neuronal apoptosis following DNA damage. Given the neuronal functions of CREB, it is conceivable that disregulation of the ATM-CREB pathway contributes to neurodegeneration in A-T.

NF-_B Super-induction: A Mechanistic Investigation on the Novel Cross-talk pathway for NF-_B Signaling

*Pei-Yun (Beth) Chang and Shigeki Miyamoto
Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin – Madison, USA, 53706

Various chemotherapeutic agents (e.g. etoposide, camptothecin, ionizing radiation) can induce NF-_B activation, which potentially underlies a mechanism of resistance for cancer treatment. The mechanism by which these agents activate NF-_B is largely unknown. Moreover, cross-talk pathways that regulate the signaling cascade remain a mystery. We discovered that NF-_B activation by several chemotherapeutic agents is augmented by prior treatment with TPA (12-myristate-13-acetate), termed “super-induction”. Super-induction correlates with decreased apoptosis in response to etoposide. Further analysis showed transcriptional activation of certain cytokines and cell cycle regulators in response to super-induction. This response was observed selectively in several malignant cell lines (e.g. CEMp, 70Z/3), but not any of the normal cell types tested (e.g. NhMSC). Interestingly, super-induction appears to be mediated through a direct modification of the NF-_B protein. The underlying mechanism and pathological importance of the cross talk of the two ubiquitous signaling cascades remained to be investigated. The long-term goal of the proposed research is to develop strategies that exploit NF-_B signaling to control the pathogenesis of carcinogenesis.

A Motor Makes Sense: Identification and Cloning of a Kinesin-Like Gene Required for Sensory Neuron Function in Male C. elegans Mating Behavior

Erik Peden
Molecular and Cellular Pharmacology Program
Laboratory of Maureen Barr, School of Pharmacy

All animals must sense and respond appropriately to the environment to survive and reproduce. The roundworm Caenorhabditis elegans, with its simple behaviors and completely sequenced genome, provides an excellent model to study the genes and molecules required for sensory transduction. We are using the male-mating behavior of C. elegans as a model sensory system to identify and characterize genes and signal transduction pathways required for complex sensory behaviors.

I am cloning and characterizing a gene required for sensory signal transduction in a subset of male mating behavior neurons. Males homozygous with the mutant allele, designated sy511, exhibit defects in two sensory steps of the mating program: response to hermaphrodite contact (i.e. fail to initiate mating behavior) and location of mates vulva, (Lov). Microscopic anatomical analysis of sy511 males reveals that the differentiation and morphologies of the neurons that regulate these steps appear normal, suggesting sensory defects of sy511 are not due to abnormal neuronal development but rather to impaired sensory transduction. Genetic (3-factor cross) and physical (SNP) mapping has placed sy511 within a 7-cosmid interval on Linkage III, and we now report transgenic rescue of the sy511 male mating defect with a single cosmid. Sequence analysis of the 10 open reading frames (ORFs) on this cosmid at WormBase (www.wormbase.org) reveals a promising candidate, klp-6, which is predicted to encode a kinesin motor-like protein.

We are determining if PCR-amplified wild-type genomic sequence of klp-6 with 1.4 kB of 5’ upstream promoter sequence will rescue sy511 mating defects. Additionally, in a sy511 background, we observe cellular mislocalization of a GFP labeled sensory channel, PKD-2:GFP, that normally localizes specifically to male sensory cilia. We hypothesize that KLP-6 participates in the trafficking of PKD-2 and possibly other membrane and/or signaling proteins along microtubules in a subset of sensory neurons. Our results will shed light on membrane protein trafficking and human diseases.