XBP1 Controls Diverse Cell Type- and Condition-Specific Transcriptional Regulatory Networks

Using genome-wide approaches, we have elucidated the regulatory circuitry governed by the XBP1 transcription factor, a key effector of the mammalian unfolded protein response (U2R), in skeletal muscle and secretory cells. We identified a core group of genes involved in constitutive maintenance of ER function in all cell types and tissue- and condition-specific targets. In addition, we identified a cadre of unexpected targets that link XBP1 to neurodegenerative and myodegenerative diseases, as well as to DNA damage and repair pathways. Remarkably, we found that XBP1 regulates functionally distinct targets through different sequence motifs. Further, we identified Mist1, a critical regulator of differentiation, as an important target of XBP1, providing an explanation for developmental defects associated with XBP1 loss of function. Our results provide a detailed picture of the regulatory roadmap governed by XBP1 in distinct cell types as well as insight into unexplored functions of XBP1.

Mice Deficient in αβ6 and αβ8 Phenocopy TGFβ1 and TGFβ3 Null Mice

The TGFβ cytokines regulate many cellular functions. Of three TGFβ isoforms, TGFβ1 is the major form expressed by leukocytes. The importance of TGFβ1 in the immune system is highlighted by the fact that TGFβ1 knockout mice develop a fatal autoimmune illness. TGFβ2- and TGFβ3-null mice die at birth (due to cardiac defects and cleft palate, respectively) and have no known immunologic deficits.

TGFβs are secreted in a latent form. We previously showed that two integrins (αβ6 and αβ8) activate latent TGFβ1 and TGFβ3 in vitro. β6-/- mice develop mild inflammation in lung and skin, and β8-/- mice are born with intracerebral hemorrhage and occasionally cleft palate. Thus, the phenotypes of β6-/- and β8-/- mice partially overlap TGFβ1- and TGFβ3-null phenotypes. To test the extent to which TGFβ1 and TGFβ3 are activated by the integrins avβ6 and avβ8 in vivo, we generated mice deficient in both avβ6 and avβ8.

We found that β6-/-;β8-/- mice uniformly develop cleft palate, suggesting that in combination these two integrins activate TGFβ3 during palate fusion.  To assess the combined role of αvβ6 and αvβ8 in immune function, we treated β8-/- mice with an inhibitory anti-αvβ6 antibody beginning after palate fusion. The consequences for immune function depended strongly on the timing of αvβ6 inhibition: antibody injections beginning 3 days after birth resulted in only mild multiorgan inflammation without lethality, whereas injections beginning several days before birth and continuing postnatally produced a lethal autoimmune syndrome identical to, if not more severe than, that seen in TGFβ1-null mice. These are the first data indicating a role for avβ8 in immune homeostasis.  The simultaneous genetic and/or pharmacological inactivation of the integrins αvβ6 and αvβ8 results in phenotypes consistent with a model in which these two integrins together are required for all TGFβ1 and TGFβ3 activation.

Molecular Mechanism of Reactivation of Herpes Simplex Virus

Neurotrophins regulate survival, differentiation, and growth of neurons and modulate synaptic plasticity.  NGF also contributes to the maintenance of latent Herpes Simplex Virus type I (HSV-1) infections in neurons. HSV-1 is neurotropic and has two potential states within neurons, lytic and latent.  While the lytic phase has been extensively studied and results in the production of infectious virus followed by the destruction of the host cell, there is little known about how HSV-1 latency is established and maintained in neurons.  Previous work by Wilcox and Johnson found that withdrawal of NGF from cultured rat sympathetic neurons resulted in the reactivation of latent HSV-1. However, the mechanism by which NGF promotes the maintenance of HSV-1 latent infections has not been defined.  NGF uses two different transmembrane receptors, p75 and TrkA, which activate downstream targets, such as PI-3K, Akt, PLCg, Ras, Raf, and Erk. I established a quiescent HSV-1 infection in sympathetic neurons, which resembles latency. This state is characterized by the presence of a latency-specific RNA transcript (LAT); the absence of HSV-1 transcripts and proteins required for lytic viral replication; and the lack of infectious viral particles as assessed by plaque assay.  Using pharmacological inhibitors, I found that inhibition of either the NGF TrkA receptor or PI-3K induced HSV-1 reactivation in sympathetic neurons. MEK1/2 inhibition did not induce reactivation or measurably disrupt the quiescent infection.  These results indicate that the ability of HSV-1 to remain in a quiescent, latent-like state in cultured rat sympathetic neurons requires continuous Trk signaling that in turn triggers downstream effectors such as PI-3K. Furthermore, they suggest that active NGF-signaling results in a environment that does not support lytic HSV-1 replication and raise the possibility that host signal transduction pathways are critical to for the maintenance of life-long latent HSV-1 infections in its human host.

Progeria, LMNA, and Insight Into Normal Aging

Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic form of premature aging. HGPS patients exhibit numerous features reminiscent of common human aging, with death occurring at a mean age of 13, usually from heart attack or stroke. HGPS is caused by a de novo point mutation in LMNA that results in production of a mutant lamin A protein, termed “progerin”. Due to its permanent farnesyl-anchor, progerin disrupts the normal architecture of the nuclear envelope, thus leading to the misshapen, blebbed nuclei that are the cellular hallmark of the disease. Here we show that inhibiting this farnesylation step pharmacologically both restores normal nuclear architecture and both prevents and reverses cardiovascular disease in a HGPS mouse model. Finally, we provide significant evidence for an association between a variant form of the LMNA gene and human longevity. These studies have both provided encouraging evidence for the initiation a clinical trial for HGPS, and have offered significant insights into possible molecular mechanisms of human aging.

Regulation of Transcriptional Elongation and Termination in Escherichia coli

The high information density of prokaryotic genomes requires that transcription terminate precisely at operon ends in order to avoid interference with neighboring transcription units. Both intrinsic and Rho-dependent termination signals are found at the ends of operons. We used chemical and genetic perturbations of Rho function in microarray gene expression experiments to assess the role of Rho and termination factors NusA and NusG in transcription regulation on a genome-wide scale. Our results indicate that inhibition of Rho affects the expression of most E. coli genes, in particular, it strongly derepresses genes encoded by prophages and recently-acquired portions of the genome. Comparison of transcriptome and proteome content during Rho inhibition shows that cryptic genes, normally repressed by Rho, do not produce protein efficiently.

Proapoptotic BIMEL Is Degraded By βTRCP Ubiquitin Ligase Complex

BimEL (Bcl-2 interacting mediator of cell death, extra long isoform)  is a proapoptotic member of the Bcl-2 protein family that links  stress induced signals to the apoptotic machinery. Induction of  survival signals such as the addition of growth factors promotes the  ubiquitylation and proteasomal degradation of BimEL in a  phosphorylation dependent manner. Despite BimEL evident importance in  determining cell fate, the cellular machinery responsible for its  degradation has not yet been identified. In this work we demonstrate  that SCFβTrcp1/2 is the ubiquitin ligase complex governing BimEL  protein levels. Binding of βTrcp1/2 to BimEL is dependent on  phosphorylation of BimEL on three serine residues found in a  consensus degron sequence similar to other βTrcp1/2 substrates. In  addition we show that phosphorylation of these residues is carried  out by RSK1. Our data links the ubiquitin ligase βTrcp1/2 and the  kinase RSK1 to the intrinsic apoptotic pathway via regulation of the  protein levels of BimEL.

Mechanisms of Atherosclerosis Regression

We report a model of atherosclerosis regression that involves transplanting an atherosclerotic arch into a wild type mouse. There, emigration of plaque foam cells occurred in a CCR7 dependent manner. In a dendritic cell line, we found that CCR7 levels increased 8X upon LXR activation by the agonist T0901317. To extend the results in vivo, we treated western-diet fed apoE-/- mice with LXR agonist. Interestingly, CCR7 was induced in foam cells and foam cell content as well as lesion area decreased by 21% and 24%, respectively. When we treated apoE-/-CCR7-/- mice with LXR agonist, foam cell content was only decreased by 9% suggesting that CCR7 mediates foam cell depletion. To address whether LXR is required for regression, we used apoE-/-, LXRα-/-apoE-/-, and LXRβ-/-apoE-/- mice as donors and wild types as recipients. Transplanted plaques from either of the double knockouts (DKOs) exhibited impaired regression as foam cell content and lesion area only decreased by 35% and 28%, respectively instead of 67% and 59% in lesions that originated from apoE-/- mice.Therefore, LXR activation promotes regression of atherosclerotic lesions which may be in a CCR7 dependent manner.

JHDM1B/FBXL10 Is a Nucleolar Protein That Represses Transcription of Ribosomal RNA Genes

Histone modifications such as lysine methylation affect chromatin structure and gene expression. Histone methylation can be reversed by the JmjC-domain family of histone demethylases. One such histone demethylase, JHDM1B, has been suggested to be a tumour suppressor. Here, we show a probable role for JHDM1B in cancer development. We show JHDM1B represses expression of ribosomal RNA genes, via the demethylation of the trimethylated lysine 4 residue of histone H3. JHDM1B also influences cell growth and proliferation, suggesting that reduced levels of JHDM1B promote tumour development.

Inhibition of the Mammalian Target of Rapamycin (TOR) Blocks Mitogen-activated Demyelination

Demyelination results from dedifferentiation of Schwann cells and the consequent breakdown of their myelin sheaths.  Although demyelination is a significant source of morbidity in nerve injuries and neuropathies, the mechanisms involved have remained elusive. Our lab has developed an experimental model of demyelination that involves addition of the Neuregulin-1 (NRG1) growth factor to myelinating cocultures.  This results in the activation of the erbB receptors and their downstream signaling pathways followed by Schwann cell dedifferentiation, proliferation, and demyelination.  Among the pathways activated by NRG1 is the mammalian target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational apparatus and integrates a number of upstream signals.  We have found that inhibiting mTOR with rapamycin effectively prevents NRG1-induced demyelination.  Intriguingly, we have also found that mTOR activity is elevated in trembler-J mice, a mouse model of the demyelinating human neuropathy Charcot-Marie-Tooth 1A.  Therefore, elucidation of the mTOR pathway may provide new insights into the mechanisms of demyelination and identify new targets for its treatment.

Knockdown of Tankyrase 1 Induces Senescence and Telomere  Dysfunction in HTC75 Cells

Tankyrase 1, a telomeric poly(ADP-ribose) polymerase, is a positive  regulator of telomere length.  Upon overexpression of tankyrase 1 in  the nucleus, tankyrase 1 poly (ADP-ribosyl)ates TRF1, removing it  from telomeres.  Loss of TRF1 results in telomere elongation,  dependent on telomerase.  Another function of tankyrase 1 was  revealed by knockdown of tankyrase 1 expression.  Tankyrase 1 siRNA  treatment in HeLa cells resulted in mitotic arrest.  Sister  chromatids were able to separate at centromeres and arms, but were  unable to separate at their telomeres.  Interestingly, tankyrase 1  siRNA in HTC75 cells (a derivative of HT1080 fibrosarcoma cells) did  not lead to mitotic arrest.  To further examine the effect of  tankyrase 1 knockdown in HTC75 cells, we used lentiviral shRNA  vectors to generate stable cell lines lacking tankyrase 1.  Knockdown  of tankyrase 1 using four different shRNAs led to a severe growth  defect.  These cells were found to be senescent by characteristic  morphology and expression of SA β-galactosidase. Furthermore,  these cells contain DNA damage foci and telomeric fusions.

A New Connection in Ras Signaling Pathway

The RTK-Ras-MAPK cascade functions as a central signaling pathway to control most of fundamental cellular processes. In response to activated RTKs, Son of Sevenless (Sos), a Ras guanine nucleotide exchange factor, is recruited to the plasma membrane by activated RTKs and subsequently activates Ras. Our structure-function analyses of Sos identified two binding pockets for Ras proteins: one is catalytic site for nucleotide exchange of Ras from GDP to GTP; the other is allosteric site for interaction with GTP-bound Ras, which leads to elevated catalytic activity of Sos and consequent Ras activity. Because oncogenic forms of Ras are "locked" in the GTP-bound state, we hypothesize that oncogenic Ras may bind to the allosteric site of Sos and as a result the activity of wild-type Ras will be increased. The goal of this project is to determine if activation of WT Ras through oncogenic Ras-mediated allosteric stimulation of Sos is required for tumorigenic properties.

Interaction Between Telomere Associated Proteins and Cohesin and Condensin

Telomeres are nucleoprotein structures composed of TTAGGG double strand DNA tandem repeats and a 3' overhang at chromosome termini. They serve critical functions in chromosome end replication and chromosome end protection. Due to DNA polymerase's inability to duplicate de novo, chromosomes lose their ends with each replication process. The loss of chromosome ends, in turn, leads to genomic instability. In conjunction with telomerase, a ribonucleoprotein reverse transcriptase, telomeres resolve this problem. In addition, they protect chromosome ends, which resemble DNA double strand breaks, from unnecessary DNA repair, checkpoint induction, and degradation.

Telomeres are bound by Shelterin, a six-subunit protein complex. Shelterin consists of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1. TRF1, double strand DNA binding protein, negatively regulates telomere length. On the other hand, a TRF1 related protein TRF2, protects telomeres. TIN2 bridges TRF1 and TRF2 to form a six-subunit complex. TPP1 negatively regulates telomere length.

Based on our previous studies, sister chromatid telomeres require special mechanisms involving cohesin and condensin to separate themselves in mitosis. We identified that Shelterin subunits TRF1 and TIN2 interact with a cohesin subunit SA1. Cohesin is a protein complex composed of SMC1, SMC3, SCC1, and SCC3 (SA1 or SA2 in mammalian cells). It holds sister chromatids from replication until segregation. We, furthermore, observed that TIN2, TPP1, and weakly TRF1 interact with a condensin subunit CAPH. Condensin (a protein consisted of SMC2, SMC4, CAPH, CAPD2, and CAPE) compacts chromatids

Roles of Abnormal Endocytosis in Pathogenesis of Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia, caused by synaptic dysfunction and neuronal death.  The endocytic pathway plays critical roles in AD pathology, including proteolysis of amyloid precursor protein (APP), generation of amyloid beta (Ab), signaling of neurotrophic factors, and synaptic plasticity.  Early endosomes become abnormally enlarged in neurons at early stage of AD, suggesting dysfunctions of the endocytic pathway. Rab5 is a small GTPase located in early endosome and involved in size and function of early endosomes.  Therefore, investigating the role of endocytosis, especially Rab5, in AD is critical to an understanding the disease.  To pursue this, a transgenic mouse overepxressing Rab5 will be used as in vivo model as well as primary neuronal cultures obtained from the mouse cortex as in vitro system.

Establishment of a 3D Culture System of Mouse Primary Pancreatic Duct Epithelial Cells (PDEC) to Study Pancreatic Tumorigenesis

Pancreatic ductal adenocarcinoma is an aggressive and devastating malignancy which shows profound resistance to treatment, and is generally thought to arise from ductal epithelium. The availability and characterization of a 3D culture system of mouse primary pancreatic duct epithelial cells (PDEC) would permit elucidation of the relationship between specific genetic lesions and the morphogenetic changes that accompany pancreatic ductal tumorigenesis. Emerging evidence suggests that either putative pancreatic stem cells or dedifferentiated derivatives of mature cell types may be involved in pancreatic tumorigenesis. Since 3D culture of PDEC may contain pancreatic progenitor cells, identification and characterization of pancreatic progenitor cells in 3D culture would be particularly useful for studying a role for pancreatic progenitor cells in pancreatic tumorigenesis. Among genetic mutations associated with pancreatic tumorigenesis, oncogenic Kras mutations are generally believed to be essential for the initiation of pancreatic cancer. We have characterized 3D culture system of mouse PDEC and have found that physiological levels of oncogenic Kras in 3D culture of PDEC increase cellular proliferation. We are currently examining whether mature PDEC and pancreatic progenitor cells have different susceptibility to oncogenic Kras to induce cellular proliferation in 3D culture of PDEC.

A Novel Role for SWI/SNF Subunit BAF250b as an E3 Ubiquitin Ligase Substrate Adaptor

The mammalian ATP-dependent chromatin remodeling complex SWI/SNF consists of the catalytic ATPase Brg1 or Brm and associated factors (BAF's). There are two isoforms of the largest subunit, BAF250a and BAF250b (or ARID1A/B), which contain the ARID DNA binding domain.  A mass spectrometry screen of BAF250b protein interactors identified elongin C and Cullin.  Elongin C and cullin2/5 are known to assemble with a substrate adaptor and the RING finger protein Roc1/Rbx1 to form an E3 ubiquitin ligase.  Elongin C binds its substrate adaptors via the BC box motif.  We have found that the C-terminus of BAF250b binds elongin C in a BC box dependent manner.  Cullin2 associates with BAF250b's C-terminus and ARID domain.  Mutating the BC box in BAF250b results in polyubiquitination and proteosome dependent degradation of BAF250b. Flag tagged BAF250b was immunopurified under stringent conditions for in vitro histone ubiquitination assays.  The discovery that the BAF250b-elongin B/C Cul2-Roc1 E3 ubiquitinates histones adds a new enzymatic function to SWI/SNF.

NSD1, A Histone Methyltransferase That Works in Ligand-Dependent Transcription

otos syndrome belongs to a group of neuroproliferative diseases associated with macrocephaly and excessive growth. Haploinsufficiency of Nuclear receptor binding SET Domain-containing protein 1 (NSD1) is the major cause of Sotos syndrome (Kurotaki et al., 2002). NSD1 binds unliganded and liganded nuclear receptors (NRs) through different NR interacting domains (NIDs) (Huang et al., 1998). These NIDs exhibit binding properties of NIDs found in NR corepressors and coactivators, which suggests that NSD1 functions as a bivalent transcriptional regulator (Huang et al., 1998). Moreover, NSD1 has been reported to be histone lysine methyltransferase specifically targeting H3K36 and H4K20 through its SET domain. The goal of this study is to explore the role that NSD1 plays in NR dependent transcriptional regulation by studying its associating proteins and HKMTase activity, and to explore the possible malfunctions of NSD1 in Sotos syndrome by characterize some NSD1mutants found in Sotos syndrome patients.

Cell To Cell Communication in Epithelium in Response To Different Modes of Cell Death

Chronic inflammation has long been suggested to constitute a risk factor for a variety of epithelial cancers. However, the molecular and cellular mechanisms that link inflammation to tumorigenesis are not well understood. Cell death by necrosis is typically associated with inflammation, in contrast with apoptotic death. Little is known about how dying cells interact with neighboring cells in an epithelial monolayer. Since intercellular communication plays a crucial role in regulating many aspects of cell survival and tumorigenesis, we are interested in understanding the signaling events that may be activated in epithelial cells that are found in proximity of dying cells. We set out to induce two distinct type of cell death: apoptosis and necrosis in cultured mammalian cells in order to better understand how exposure to apoptotic of necrotic cells affects signaling in neighboring cells.

Pyrophosphate Removal in Eschericia coli RNA Degradation

Bacteria live in a constantly changing environment, and therefore have a continuous need to modulate their protein expression.  Three processes control protein synthesis in those microorganisms: transcription, translation, and mRNA degradation.  It had long been thought that mRNA degradation in Eschericia coli begins with endonucleolytic cleavage by RNase E. However, recent work has shown that RNase E cleavage can be triggered by a prior step: the removal of pyrophosphate from the 5' terminus of primary transcripts. An enzyme that catalyzes this initial event (RppH) has been discovered in E. coli.  Pyrophosphate removal by RppH is impeded when the 5' end is base paired, a finding consistent with previous evidence that a 5'-terminal stem-loop can increase mRNA lifetimes in bacteria.  The goal of my project is to characterize the substrate specificity of RppH and to examine its influence on the function of small regulatory RNAs.

Nucleolin Phosphorylation During Genotoxic Stress

Nucleolin (or C23) is a nucleolar protein involved in various functions such as ribosome biogenesis and cellular stress. It is phosphorylated during interphase by casein kinase 2 (CK2). Nucleolin phosphorylation by CK2 has been correlated with active rRNA transcription. My research is focused on studying the effects of phosphorylation by CK2 on nucleolin's activities in ribosome biogenesis and identifying the cellular conditions in which phosphorylation by CK2 is regulated.

Pancreatic Injury in Response to Dietary Challenge and Hyperlipidemia - A New Path to Inflammation?

Pancreatic cancer is the fourth leading cause of cancer related death in the United States and is almost uniformly fatal. Obesity is estimated to affect 33% of the adult population in the United States and recent epidemiological studies have shown that obesity is associated with an increased risk of pancreatic cancer. Given the high incidence of obesity and its association with pancreatic cancer, it is imperative to determine the mechanistic link between these disease entities.

To model the effects of obesity and consequent hyperlipidemia on pancreatic disease, we set out to establish a mouse model of hyperlipidemia driven pancreatic injury using Apolipoprotein E deficient mice (APOE-/-). APOE-/- mice have a profound defect in intermediate density lipoprotein and chylomicron clearance that leads to hypercholesterolemia and hypertryglyceridemia when placed on a high fat diet (HFD). These mice provide a more accurate model of the elevated lipid levels frequently found in obese humans. In order to determine the consequences of prolonged hyperlipidemia on the pancreas, we examined the effect of HFD on the pancreata of APOE-/- mice. Our studies demonstrate that HFD causes a mild inflammation

Potential Interactions Between Necl4 and Par3 During Myelination

Myelinated axons are organized into specialized regions, including nodes and internodes, which are crucial for proper saltatory conduction.  The molecular compositions of those regions consist of distinct complexes of cell adhesion molecules and ion channels linked by scaffolding proteins. Nectin-like proteins are cell adhesion molecules that mediate axo-glial interactions along the internode. A member of this family of proteins, Nectin-like 4 (Necl4) is expressed by myelinating Schwann cells and was recently shown to be essential for their myelination of axons in the peripheral nervous system (PNS). Necl-4 is a type I transmembrane protein that contains 3 extracellular Ig-like domains and two cytosolic binding domains, protein 4.1-ezrin-radixin-moesin (FERM) and PSD-95/Dlg/ZO-1 (PDZ) that could potentially play a role in myelination. The aim of my project is to study the function of these binding domains and their interactors in myelination. Using shRNA lentiviral constructs to knock down endogenous levels of Necl4 in Schwann cells, we found that myelination is inhibited in a co-culture system of Schwann cells and DRG neurons. To understand the function of these cytoplasmic domains of Necl4, we have generated a lentivirus expressing a recombinant form of Necl-4 containing a deletion in its PDZ binding domain (Necl4deltaPDZ). We are examining the effect of this chimeric protein on myelination by Schwann cells in cocultures to determine if it has a dominant negative effect. A molecule that is a candidate to interact with Necl4 is Partitioning-defective 3 (Par3). Par3, which has three PDZ domains, is a polarity protein recently shown to be essential for myelination in the PNS.  Knockdown of Par3 expression in Schwann cells inhibits myelination, phenocopying the effect of Necl4 knockdown, suggesting that Par3 might be a downstream effector of Necl4. To explore this possibility, we performed clustering experiments between Par3 and Necl4. Initial observations suggest a partial co-location between Necl4 and Par3. Initial biochemical analysis also supports an interaction between these proteins. These results suggest that Par3 and Necl4 interactions might be relevant for myelination.

RESEARCH TITLE AND SUMMARY NOT AVAILABLE

The Effect of the Tumor Microenvironment on Protein Synthesis

It is well established that the extracellular matrix (ECM) modulates critical tumor cell processes including survival, differentiation and angiogenesis. We asked whether the ECM also regulates protein synthesis in tumor cells. During hypoxia, tumor cell protein synthesis is generally down-regulated, impairing tumor growth and proliferation. The regulation of translation by integrins through the Akt/mTOR/4E-BP1 pathway represents a pivotal process that impacts on tumor cell responses to hypoxia, invasion and progression. We demonstrate that the integrin avb3, which is often elevated on breast tumor cells and promotes migration and invasion, interacts with vitronectin, an ECM component only available to tumors with invasive disruption of the basement membrane, resulting in sustained and high levels of protein synthesis and VEGF production despite hypoxia, through the constitutive activation of the mTOR/4E-BP1 pathway.

Signaling Through MT1-MMP

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane proteinase with an extracellular proteolytic domain and a short cytoplasmic tail. The proteolytic activity of MT1-MMP is inhibited physiologically by tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). TIMP-2 binds to MT1-MMP and the complex is internalized by endocytosis mediated by the cytoplasmic tail of the proteinase. Our extensive preliminary work in tumor epithelial cells has shown that binding of TIMP-2 to MT1-MMP rapidly induces ERK1/2 MAP kinase activation, which upregulates cell proliferation and migration. These effects require the cytoplasmic tail but not the proteolytic activity of MT1-MMP.  We have also shown that TIMP-2 addition to wild-type but not MT1-MMP knockout mouse embryonic fibroblasts (MEFs) results in ERK activation, suggesting that this mechanism is active in primary stromal cells.  Moreover, we have characterized these MEFs and shown that the absence of MT1-MMP can affect various properties including MAP Kinase Activation, cell proliferation, migration, attachment and size.

The Role of ClpP in the Mammalian Mitochondrial Unfolded Protein Response

The maintenance of appropriate mitochondrial chaperone levels is critical for folding newly synthesized and imported proteins as well as for preventing aggregation of misfolded proteins under stress conditions.  While much is understood about how chaperone levels in the cytosol and endoplasmic reticulum are regulated in response to stress, there has been less inquiry into mechanisms of chaperone regulation in mammalian mitochondria.  In C. elegans, the activity of the mitochondrial protease ClpP is necessary for signaling from the mitochondria to the nucleus that results in a transcriptional response to misfolded protein stress, including upregulation of chaperones. We hypothesize that ClpP initiates signaling of mitochondrial stress in mammalian cells by degrading substrates in a stress-specific manner.   Our aim is to elucidate the mechanism of ClpP signaling via identification of partners and substrates, enabling further investigation of the regulation, targets, and significance of this signaling pathway.

Regulation of the Drosophila Apoptosome by Degradation

The Drosophila Apoptosome is a cell-death triggering holoenzyme formed by the Drosophila Apaf-1 homolog and the Drosophila initiator caspase, Dronc. Dronc has been previously shown to be a target of ubiquitylation by the E3 ubiquitin ligase Diap (Drosophila inhibitor of apoptosis). Preliminary in vivo data suggests that Apaf-1 and Dronc are not only coupled in activation, but negatively regulate each other's protein level.  I plan to study the mechanism (or mechanisms) underlying the regulation of the Drosophila Apoptosome, and how it is involved in the regulation of apoptosis. To achieve this goal, I will use in vitro assays (binding and ubiquitylation), S2 cell culture assays (protein half-life and co-immunoprecipitation), and in vivo assays (examination of proteins levels in Drosophila tissues). Using these assays, I will perform a screen to identify factors that are involved in the regulation of Apoptosome stability.

Characterization of Phospho-K-Ras Induced Cell Death

K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We have shown that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, where phospho-K-Ras interacts with Bcl-XL.  PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner.  The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion.  K-Ras with a phosphomimetic residue at position 181 induces caspase activation via a pathway that requires Bcl-XL.  Artificial targeting of K-Ras to endoplasmic reticulum (ER) and mitochondria pinpointed the ER as the source of the apoptotic signal.  This signal does not appear to propagate through the canonical cell death pathways and may instead utilize an ER calcium-mediated apoptotic pathway. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate this ER-specific cell death pathway.

β-Catenin Signaling in Mammary Development and Tumorigenesis

Recent evidence has shown that breast tumors arise from, and can be propagated by, a small percentage of tumor initiating cells . β-catenin signaling is deregulated in human breast cancer and in several other human cancers including colon cancer, skin cancer and leukemia. Significantly, in the latter three tissues, β-catenin has been shown to accompany and regulate stem/progenitor cell proliferation and maintenance. Canonical Wnt signaling is essential for embryonic and adult mammary development. Our laboratory has generated a transgenic mouse that expresses a stabilized form of β-catenin (MMTV-ΔN89β-catenin). These mice develop mammary adenocarcinomas at the early age of 4.5 months. Development of these tumors is preceded by precocious alveolar development, suggesting a connection between activation of pathways controlling alveologenesis and predisposition to breast cancer. Using mice carrying reporters of β-catenin signaling, I have identified β-catenin signaling cells in the glands and tumors of MMTV-ΔN89β-catenin mice. A subset of luminal cells undergo β-catenin signaling in MMTV-ΔN89β-catenin glands and give rise to mammary hyperplasias, structures from which the tumors are thought to arise. The majority of β-catenin signaling cells in MMTV-ΔN89β-catenin tumors fall in a CD24(+)CD49f(low) population and reflect the Sca-1 characteristics of total tumor cells. A subpopulation of β-catenin signaling cells, however, form part of a CD24(med)CD49f(high) population which, in the normal gland, has been reported to contain potential mammary stem cells, otherwise known as Mammary Replicating Units. These results suggest that mammary tumors caused by deregulated β-catenin signaling may arise from a subpopulation of tumor initiating cells that could be considered as “cancer stem cells.

Identification and Characterization of a p56lck-Interacting Protein Expressed in CD8+ Tumor Infiltrating TCcells

Immune response to cancer has been long recognized, including both innate and adaptive responses, showing that the immune system can recognize protein products of genetic and epigenetic changes in transformed cells. However, cancers grow, implying that antitumor immune responses are either not sufficiently vigorous to eliminate the tumor or that antitumor immunity is suppressed (a form of immunological tolerance). Inhibition of specific antitumor immunity is common and several possible explanations of tolerance to tumor antigens or tumor-induced immunesuppression have been proposed. Inhibition of effective antitumor immunity results from inhibition of the activation, differentiation, or function of antitumor immune cells due to tumor growth or the host response to tumor growth. As a consequence, antitumor T cells cannot respond productively to developmental, targeting, or activation cues.

Our lab studies the phenotype of CD8+ antitumor T cells that infiltrate tumor tissue ('TIL') wherein we have shown that lytic function of CD8+ antitumor TIL is defective and is correlated with blockade of proximal TCR-mediated signal transduction. When purified and briefly cultured in the absence of tumor cells TIL recover both proximal signaling and lytic function. This suggests the involvement of a fast acting ‘switch’ in the regulation of TIL function and also that the inhibitory signal is tumor-derived. Upon conjugation in vitro with cognate tumor cells freshly-isolated TIL initiate signaling but which is abrogated such that ZAP70 is not activated. Since the T cell src family kinase p56lck controls activation of ZAP70 (and therein all downstream signaling events) we have analyzed the function of p56lck and determined that p56lck kinase function is inactivated by rapid contact-dependent dephosphorylation of the p56lck activation motif (by Shp-1) thus accounting for both defective signal transduction and effector phase function of TIL.

In the course of those studies we observed a ~115Kd protein which co-immuneprecipitates with p56lck from TIL detergent lysates. Since the 115Kd protein was observed by reciprocal immunoblotting using an antipeptide antibody reactive to an epitope contained in p56lck, we conducted a BLAST analysis using the peptide motif (EGQYQPQP) to search the protein database and tentatively identify the 115Kd protein as Protocadherin-18 (Pcdh-18), a member of a multigene family of adhesion molecules. This protein is the only protocadherin isoform and also the only non-src kinase that contains that peptide motif. Thus, serendipitously we have identified an interacting partner for p56lck, the T cell src kinase, and tentatively determined its identity.

We hypothesize that tumor-induced defective p56lck activity in CD8+ tumor infiltrating T cells may be mediated by a novel p56lck-binding protein, Protocadherin-18.

Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1.  The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles.  Exocytosis of these vesicles delivers Rap1 to the plasma membrane where it becomes activated.  We now show that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as is Rap1 and is translocated to the plasma membrane along with Rap1.  Moreover, PLD activity is required for both translocation and activation of Rap1.  Increased T cell adhesion in response to stimulation of the antigen receptor also depended on PLD1.  C3G, a Rap1 guanine nucleotide exchange factor (GEF), also translocates to the plasma membrane in stimulated T cells.  Our data support a model wherein PLD1 regulates Rap1 activity by controlling exocytosis of a stored pool of vesicular, inactive Rap1 that upon delivery to the plasma membrane, can be activated by C3G.

Regulation of SOS by its Histones-Fold Domain - Lessons from Noonan Syndrome

The duration and intensity of RTK signaling determines various cellular outcomes including survival, proliferation, differentiation, vesicular trafficking and cytoskeleton reorganization. SOS, a Guanine nucleotide Exchange Factor (GEF), couples the RTKs to the Ras-MAPK pathway by mediating signal dependent activation of Ras. A complex interplay of incompletely understood inter/intra-molecular interactions regulates SOS function. Recently we reported a panel of gain-of-function SOS mutations, causing Noonan Syndrome (NS) disease. SOS is also a GEF of Rac, which controls cytoskeletal rearrangement. Here, we show that these mutations also cause hyper activation of Rac1.

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