Abstract The metabolic syndrome is a cluster of components predisposing to type 2 diabetes and cardiovascular disease. Abdominal obesity and insulin resistance seem to be central in the metabolic syndrome, although no unifying pathophysiological mechanism is available. The aim of this thesis was to determine out how the variation in PTP1B and adiponectin gene as well as variations in the plasma adiponectin concentration contribute to the risk of obesity related diseases. PTP1B is a negative regulator of insulin signalling and therefore considered a candidate gene for type 2 diabetes. In the first study, it was found that three PTP1B polymorphisms studied have not strong impact on type 2 diabetes. However, one SNP may be slightly protective against type 2 diabetes, since it was more frequent in the healthy group compared to group of patients with type 2 diabetes. Another SNP was associated with body mass index (BMI). The combination of certain alleles of PTP1B and LEPR (leptin receptor) genes was also associated to BMI. Adiponectin is an adipocytokine expressed in adipose tissue. It has insulin sensitizing effects in liver and muscle and it has also beneficial effects on cardiovascular health. In the second study, the contribution of adiponectin genotypes with obesity-related phenotypes was studied. In Caucasians, the carriers of rare allele of Tyr111His polymorphism were more insulin resistant and at a higher risk of developing type 2 diabetes. In African-Americans, other polymorphisms were associated with BMI and lipids. Thus, the effects of polymorphisms on obesity related phenotypes seemed to be different between ethnic groups. Plasma adiponectin levels were measured from different study groups. In the third study, it was found out that low plasma adiponectin levels associated with different components of the metabolic syndrome and there was a trend towards reductions in adiponectin with an increasing number of components. Fourth study indicated that baseline low adiponectin level associated with a more than 2-fold risk for developing impaired glucose tolerance or type 2 diabetes in the follow-up study of normoglycemic middle-aged Finnish subjects. In the fifth study, plasma adiponectin levels were measured from postmenopausal women receiving estrogen replacement therapy. We observed a reduction in adiponectin levels in women having peroral estradiol which could be part of the "early harm" profile on cardiovascular risk factors of the peroral estrogen replacement therapy detected in clinical trials. These studies further strengthen the role of plasma adiponectin in the obesity related diseases and bring new information of polymorphisms in the adiponectin and PTP1B genes in different populations. / Tiivistelmä Metabolinen oireyhtymä on kertymä tekijöitä, jotka altistavat tyypin 2 diabetekselle ja sydän- ja verisuonitaudeille. Keskivartalolihavuus ja insuliiniresistenssi, eli insuliinin heikentynyt teho, vaikuttavat olevan keskeisiä metabolisessa oireyhtymässä. Kuitenkaan taustalla olevaa syntymekanismia ei täysin tunneta. Väitöskirjatyön tavoitteena oli tutkia PTP1B- ja adiponektiinigeenin muuntelun sekä plasman adiponektiinitason yhteyttä metaboliseen oireyhtymään, sen osatekijöihin ja seurauksiin. PTP1B on insuliinin toimintaa soluissa estävä molekyyli. Ensimmäisessä tutkimuksessa havaittiin että kolme tutkittua PTP1B-geenin nukleotidimuutosta eivät ole vahvasti yhteydessä tyypin 2 diabetekseen. Eräs nukleotidimuutos saattaisi olla lievästi suojaava tyypin 2 diabetesta vastaan, sillä se oli yleisempi terveillä kuin tyypin 2 diabetesta sairastavilla. PTP1B:n ja leptiinireseptorigeenin eräiden alleelien yhdistelmä oli yhteydessä painoindeksiin. Adiponektiini on rasvakudoksen erittämä hormoni, jolla on suotuisia, insuliinin vaikutusta edesauttavia vaikutuksia elimistössä sekä edullisia vaikutuksia verenkiertoelimistössä. Toisessa työssä havaittiin että Amerikan valkoihoisilla, joilla oli eräs harvinainen adiponektiinigeenin alleeli (Tyr111His), oli heikompi insuliinin teho kuin henkilöillä joilla ei ollut kyseistä muutosta. Tämä alleeli oli yleisempi suomalaisilla tyypin 2 diabetesta sairastavilla kuin terveillä, mikä saattaa tarkoittaa että se liittyy suurentuneeseen riskiin tyypin 2 diabetekselle. Afroamerikkalaisilla taas toiset nukleotidimuutokset olivat yhteydessä lihavuuteen ja plasman rasva-arvoihin. Adiponektiinin pitoisuutta plasmassa mitattiin erilaisissa aineistoissa. Kolmannessa tutkimuksessa havaittiin, että matala pitoisuus oli yhteydessä metabolisen oireyhtymän eri osatekijöihin ja pitoisuus oli sitä matalampi, mitä enemmän osatekijöitä henkilöllä on. Neljännessä tutkimuksessa havaittiin että matala plasman adiponektiinipitoisuus oli yhteydessä suurentuneeseen riskiin saada huonontunut glukoosin sietokyky tai tyypin 2 diabetes tulevaisuudessa. Viidennessä tutkimuksessa adiponektiinitaso määritettiin naisilta jotka olivat ohittaneet vaihdevuodet ja saivat estrogeenikorvaushoitoa. Havaittiin että plasman adiponektiinitaso laski niillä naisilla, jotka saivat korvaushoitoa suun kautta. Tämä saattaisi osittain selittää suun kautta annettavan estrogeenikorvaushoidon epäedullista vaikutusta sydän ja -verisuonitautien riskitekijöihin. Tutkimus vahvistaa edelleen adiponektiinin merkitystä lihavuuteen liittyvissä sairauksissa ja tuo uutta tietoa adiponektiini- ja PTP1B-geenien muuntelun merkityksestä eri väestöissä.

Regulation of transcription in prokaryotes is primarily governed at the transcription initiation step. This feature has been extensively characterized in model prokaryotes notably Escherichia coli and Bacillus subtilis. Transcription initiation was initially thought to be governed primarily by initiation factors that recruit the RNA polymerase (RNAP) enzyme to initiate expression of given gene. Recent studies reveal multiple mechanisms at play including additional protein factors that can modulate gene expression. Nonetheless, understanding transcription factors is key to rationalize the nuanced changes in prokaryotic gene expression in response to diverse environmental stimuli. This is particularly relevant in the case of the human pathogen, Mycobacterium tuberculosis, especially due to the ability of this bacterium to survive in the host, often for several decades prior to the onset of the disease. Transcription initiation factors, also called σ factors in prokaryotes, are diverse in size and sensory/regulatory mechanisms. Indeed, the number of alternate σ factors vary substantially from six in E. coli to more than 118 in Plesiocystis pacifica. The large number of alternative σ factors has been suggested to be correlated with the diversity of micro-environments experienced by a bacterial cell. Studies on several prokaryotic σ factors reveal common features in these proteins that was not evident earlier due to poor sequence conservation. A central theme that emerges from these studies is that a minimalistic architecture of two domains can recognize promoter DNA and recruit the RNAP enzyme to initiate transcription. Additional domains are required when certain promoter elements are missing or to enable a specific, context dependent regulatory mechanism. The work reported in this thesis was influenced by previous studies in this laboratory and elsewhere on M. tuberculosis σ factors. While these studies revealed multiple features of transcription initiation, several aspects of this mechanism, including some classes of σ factors remain to be examined. The focus of this study was to examine an under-explored sub-group of σ factors, classified as the ECF41 sub-group. This sub-group has an additional domain at the Carboxy-terminus that has been hypothesised to influence σ factor activity. Towards this goal, M. tuberculosis σJ was examined. Previous studies suggested a role for this σ factor in modulating the response to hydrogen peroxide stress. An intriguing feature based on sequence analysis was that neither did this extra-cytoplasmic function σ factor have an anti-σ factor that can respond to oxidative stress nor was it directly associated with a mechanism to sense oxidative stress. The specific goal of the research described here was to understand the structural and mechanistic features that govern σJ activity. This thesis is organized as follows- The first chapter provides a brief introduction to prokaryotic transcription and regulatory mechanisms that govern this process. This chapter also has the literature necessary to phrase the problem in characterizing this family of proteins with particular reference to the unique physiology of Mycobacterium tuberculosis. A summary of the previous work is provided in this chapter to place the current study in context of previous studies and highlight the lacunae in our understanding of the transcription mechanism in M. tuberculosis. Chapter two describes the structural characterization of M. tuberculosis σJ by single-crystal X-ray diffraction. The poor sequence similarity of σJ to known σ factors precluded efforts to obtain phase information by molecular replacement methods. Here we also describe the steps that were essential to obtain diffraction quality crystals and the subsequent steps to account for pseudo-merohedral twinning, an imperfection that could have potentially been a limitation for structure determination. The crystal structure of σJ provide an example of successful phase determination with data collected on near-perfectly twinned crystals using single-wavelength anomalous dispersion. Chapter three describes computational efforts to understand the regulatory mechanisms of M. tuberculosis σJ. Classical Molecular Dynamics (MD) simulations were performed to understand the role of a C-terminal SnoaL_2 domain in this transcription factor. The MD simulations suggest that the C-terminal SnoaL_2 domain limits inter-domain movements between σJ2 (the pribnow box binding domain) and σJ4 (the -35 promoter element binding domain) and confers a compact three domain organization to this protein. The biochemical and functional characterization of M. tuberculosis σJ is described in chapter four. This includes in vitro studies on σJ and cognate promoter DNA interactions performed using Surface Plasmon Resonance (SPR) and Electrophoretic Mobility Shift Assays (EMSA). The ex vivo reporter based experiments to examine the effect of SnoaL_2 domain on σJ activity are also described. Spectroscopic studies on σJ interactions with a small molecule limonene-1,2-epoxide suggested a potential novel role for the SnoaL_2 domain in σJ. Chapter five summarizes the work on M. tuberculosis σJ reported in this thesis. We note that this study opens up a new perspective to understand σ factors. In particular, M. tuberculosis σJ suggests that the domain organization is likely to be retained in ECF41 sub-group of σ factors. This study also hints at broader implications in the distinction between one-component systems and transcription factors. Bioinformatic analysis suggest that observations similar to that noted in M. tuberculosis σJ are likely to be more widespread across diverse phyla than currently acknowledged. This thesis has three annexures. Annexure-I summarizes experimental details of the work performed on the M. tuberculosis σ/anti-σ factor complex σH/RshA. Annexure-II summarizes experimental details and strategies that could not be incorporated in the main body of this thesis. Annexure-III describes a short project performed on a bi-domain protein tyrosine phosphatase PTP99A.

Afin d'apporter une aide pour la chimiotaxonomie du genre Stereocaulon, les acides gras totaux de dix espèces de Stéréocaulon, de quelques cyanolichens et d'une cyanobactérie ont été étudiés. Les profils obtenus des acides gras saturés, insaturés et ramifiés ont été comparés par classification hiérarchique ascendante. Les acides gras iso et antéiso pourraient permettre de distinguer le genre Stereocaulon des cyanolichens à la différence des autres classes d'acides gras. Dans un deuxième temps, l'étude phytochimique du lichen S. evolutum récolté en Bretagne a permis d'isoler et de caractériser 22 métabolites secondaires. Parmi ces composés, deux sont nouveaux. Sept molécules possèdent une structure proche de l'atranorine, depside abondant et récurrent chez les Stéréocaulons. L'atranorine ayant montré lors d'un criblage une activité intéressante sur le virus de l'hépatite C, six molécules ainsi que deux dérivés hémisynthétiques ont alors été évalués. L'atranorine et le 4-O-déméthylbarbatate de méthyle ont présenté une bonne activité anti-VHC, le premier ciblant l'étape de pénétration et le deuxième l'étape de réplication. D'autres composés, deux depsidones et cinq diphényléthers, ont été testés sur l'enzyme Protéine Tyrosine Phosphatase 1B (PTP1B) qui est une cible d'intétêt dans le traitement du diabète de type 2 et le cancer du sein. Les deux depsidones (acide lobarique, acide norlobarique) et un diphényléther, l'anhydro-sakisacaulon A, ont montré une inhibition de PTP1B avec des CI₅₀ similaires au témoin acide ursolique. La modélisation de cette enzyme ayant été réalisée en parallèle, les études de docking ont permis de proposer des mécanismes d'interactions mis en jeu lors de l'inhibition de PTP1B par ces métabolites secondaires lichéniques et d'envisager l'optimisation structurale de ces composés en vue d'augmenter leur activité. / Total fatty acids (FA) of ten species of Stereocaulon in addition to cyanolichens and one cyanobacterium were studied. FA profiles based on saturated-, unsaturated- and branched chain-FAs were compared using a hierarchical ascendant classification. Cyanolichens differed from the Stereocaulon genus according iso- and anteiso-FAs profiles while no differences were seen according the other FAs families. The phytochemical study of Stereocaulon evolutum, harvested in Brittany, led to isolation and identification of 22 secondary metabolites. Two were new for lichens. Among the isolated compounds, seven compounds were structurally close to atranorin, a common and abundant depside in the Stereocaulon genus. In a previous screening, atranorin exhibited a noteworthy HCV inhibition, thus six lichen compounds and two synthetic molecules were also tested. Atranorin and methyl 4-O-demethylbarbatate were the most potent without any cytotoxicity, the first being active towards the virus penetration and the latter against the virus replication. Two depsidones and five diphenylethers were also isolated and were evaluated on the Protein Tyrosine Phosphatase 1B (PTP1B), a promising target for type 2 mellitus diabetis and breast cancer. Both depsidones (lobaric acid, norlobaric acid) and the diphenylether anhydrosakisacaulon A were as potent as the positive control ursolic acid. Simultaneously, a molecular docking study between PTP1B and the seven compounds suggested the most significant interactions to conceive possible more active compounds.

Indiana University-Purdue University Indianapolis (IUPUI) / The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML.

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.

Indiana University-Purdue University Indianapolis (IUPUI) / Autonomic innervation of the heart begins in utero and continues during the neonatal phase of life. A balance between the sympathetic and parasympathetic arms of the autonomic nervous system is required to regulate heart rate as well as the force of each contraction. Our lab studies the development of sympathetic innervation of the early postnatal heart in a conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2). Shp2 is a ubiquitously expressed non-receptor phosphatase involved in a variety of cellular functions including survival, proliferation, and differentiation. We targeted Shp2 in post-migratory neural crest (NC) lineages using our novel Periostin-Cre. This resulted in a fully penetrant mouse model of diminished cardiac sympathetic innervation and concomitant bradycardia that progressively worsen. Shp2 is thought to mediate its basic cellular functions through a plethora of signaling cascades including extracellular signal-regulated kinases (ERK) 1 and 2. We hypothesize that abrogation of downstream ERK1/2 signaling in NC lineages is primarily responsible for the failed sympathetic innervation phenotype observed in our mouse model. Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross structural cardiac anomalies; however, in vivo electrocardiogram (ECG) characterization revealed sinus bradycardia that develops as the Shp2 cKO ages. Significantly, 100% of Shp2 cKOs die within 3 weeks after birth. Characterization of the expression pattern of the sympathetic nerve marker tyrosine hydroxylase (TH) revealed a loss of functional sympathetic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Shp2 cKOs exhibit lineage-specific suppression of activated pERK1/2 signaling, but not of other downstream targets of Shp2 such as pAKT (phosphorylated-Protein kinase B). Interestingly, restoration of pERK signaling via lineage-specific expression of constitutively active MEK1 (Mitogen-activated protein kinase kinase1) rescued TH-positive cardiac innervation as well as heart rate. These data suggest that the diminished sympathetic cardiac innervation and the resulting ECG abnormalities are a result of decreased pERK signaling in post-migratory NC lineages.

Indiana University-Purdue University Indianapolis (IUPUI) / Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.