Alcohol affects many neurochemical processes, causing long-lasting changes in both the adult and developing brain. The Jak-Stat transcriptional activation pathway plays a role in the control of neuronal proliferation, survival and differentiation, but the effects of ethanol on the system have not been fully elucidated. The goal of this project was to define the effects of acute and subchronic ethanol exposure on the expression of proteins in the Jak-Stat pathway, using cultured NG108-15 cells, and in addition, to test the hypothesis that these effects are mediated through the NMDA receptor. I found that ethanol dose-dependently decreased Jak2 and Stat3 following subchronic exposure of NG108-15 in culture. Acute ethanol exposure caused a dose-dependent decrease in Stat3 protein levels. Incubation with MK-801 or ketamine, two noncompetitive NMDA receptor antagonists, or the receptor agonist NMDA, produced dose-dependent decreases in Stat3 protein as well.

<p>The Abl family of protein tyrosine kinases regulates diverse cellular processes by coordinating cytoskeletal rearrangements. Recent data indicate that pharmacological inhibition of Abl kinases reduces inflammation in preclinical models and in the clinic. While a previous role for Abl kinases in lymphocytes had been described, it remained unclear if Abl kinases regulate innate immune function. To explore this possibility, we generated a myeloid-specific conditional Abl knockout mouse. Using a combination of molecular, genetic, and pharmacological approaches, we demonstrate a role for Abl kinases in regulating the efficiency of macrophage phagocytosis and inflammatory responses. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Abl kinases are present at the phagocytic cup and are activated by Fcgamma receptor engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the Syk kinase. Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to Fcgamma receptor ligation. The link between Abl family kinases and Syk may be direct as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis.</p><p>Our studies also revealed a role for Abl kinases in macrophage and cancer cell invasion. Inhibition of Abl kinases suppressed cell invasion in vitro, whereas overexpression of Abl kinases enhanced extracellular matrix degradation. We found that partial loss of Abl kinase expression in myeloid cells reduced macrophage infiltration into tumors in a mouse model of breast cancer. Furthermore, pharmacological inhibition of Abl kinases reduced myeloid cell infiltration and slowed tumor growth in subcutaneous tumor models. We also found that Abl expression and activity are elevated in subsets of human tumor samples. Taken together, our results suggest Abl kinases have an important role in cancer and inflammation, and represent important therapeutic targets for their treatment.</p> / Dissertation

The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.

The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.

The Epstein-Barr virus (EBV) protein, Latent Membrane Protein 2A (LMP2A), is critical for maintaining viral latency and provides pro-survival and pro-migratory signals to EBV-positive B and epithelial cell malignancies. The N-terminus of LMP2A contains several protein-protein interaction motifs involved in the recruitment of cellular signalling proteins and it is through the recruitment of these proteins that LMP2A is able to initiate signalling. In B lymphocytes, LMP2A's ability to initiate signalling was originally proposed to proceed via a two step mechanism. Firstly, recruitment of the Lyn tyrosine kinase to the tyrosine phosphorylated YEEA site in LMP2A allows for tyrosine phosphorylation of the LMP2A immunoreceptor tyrosine-based activation motif (ITAM). This, in turn, facilitates the recruitment and activation of Syk tyrosine kinase which then initiates downstream signalling events. However, recent findings suggest this model may not be correct and argue that Syk recruitment to LMP2A is independent of the YEEA site. Therefore, we undertook a series of experiments to better understand the role of the YEEA motif and Lyn in the initiation of LMP2A signalling in B lymphocytes. We found that the YEEA site was not absolutely required for tyrosine phosphorylation of the LMP2A ITAM, or for LMP2A to activate Syk. Using siRNA to silence Lyn expression in LCLs. we found that reducing Lyn expression inhibited the ability of LMP2A to promote Syk tyrosine phosphorylation. In contrast, DG75 B cells or Lyn-deficient DT40 B cells transiently expressing higher levels of LMP2A did not require Lyn for LMP2A-mediated Syk phosphorylation. Furthermore, Lyn was not required for LMP2A-mediated Akt activation in DG75 B cells, but rather Akt activation was significantly enhanced in LMP2A-expressing cells where Lyn was reduced by siRNA. We propose that Lyn negatively regulates LMP2A-mediated Akt activation by phosphorylating Syk on Y323, which serves to recruit the c-Cbl E3 ubiquitin ligase to Syk and targets Syk for ubiquitin-mediated degradation. In sum, this work provides novel insight into how LMP2A uses Lyn to initiate and titre signalling in B cells and brings to light an unappreciated role for Lyn as a negative regulator of LMP2A-mediated Akt activation.

Social stress is a prevalent factor in society that can cause or exacerbate neuropsychiatric disorders including depression and posttraumatic stress disorder. According to the National Institutes of Health, 6.9% of adults in this country currently suffer from depression, and 4.1% suffer from an anxiety disorder. Unfortunately, current treatments are ineffective in reducing or alleviating symptoms in a majority of these patients. Thus, it is critical to understand how social stress changes in brain and behavior so that we might develop alternative treatments. Brain derived neurotrophic factor (BDNF), which binds to tyrosine kinase B (TrkB) receptors, plays a role in fear learning and in behavioral responses to stress, although we do not currently know whether BDNF promotes or prevents these responses. The purpose of this project was to understand how BDNF alters brain and behavior in response to social stress using a model of social stress in Syrian hamsters, termed conditioned defeat (CD). CD refers to the marked increase in submissive and defensive behavior following social defeat. Specific Aim (SA) 1 tested the hypothesis that BDNF, via TrkB receptors, promotes CD learning. Instead, we found that BDNF and a selective TrkB receptor agonist reduced CD and that a TrkB receptor antagonist enhanced CD. SA 2 tested the hypothesis that the behavioral response observed following systemic administration of TrkB-active drugs is mediated via their action in specific nodes of the neural circuit underlying CD. Unfortunately, the vehicle in which these drugs are dissolved independently activates immediate early gene expression making interpretation of these data impossible. Finally, SA 3 tested the hypothesis that BDNF alters defeat-induced neural activation at least in part by acting in the medial prefrontal cortex (mPFC). We demonstrated that BNDF microinjected into the mPFC site-specifically altered defeat-induced neural activation in the CD neural circuit supporting this hypothesis. Overall, these data suggest that BDNF acts to prevent social stress-induced changes in behavior, at least in part via the basolateral amygdala and the mPFC, and that BDNF-active drugs might be a useful avenue to pursue to discover new treatments for patients that suffer from stress-related neuropsychiatric disorders.

Adaptor proteins play a vital role in the propagation of cellular signals. Although they lack endogenous catalytic activity, they contain a variety of protein binding modules, which enable them to promote specific and efficient interactions with their binding partners. They form integrative platforms for a variety of molecules (e.g. lipids, tyrosine kinases, cytoskeletal and signaling proteins), and thereby link and coordinate key functions such as cell growth, motility and shape determination. Our laboratory has recently cloned a novel, 130 kDa adaptor protein, named XB130, as a structural homolog of actin-filament-associated-protein (AFAP-110), a stress fiber-binding Src substrate. However, the molecular interactions and functions of this novel adaptor remained to be elucidated. To characterize the function of XB130 we asked two general questions: (1) Is XB130 involved in the signal transduction pathways of tyrosine kinases? And (2) Is XB130 capable of regulating the cytoskeleton and/or is it regulated by the cytoskeleton? To address these questions first we investigated the tissue distribution of XB130 and discovered that it is abundantly expressed in thyroid. Therefore we asked whether it is a target of the thyroid-specific tyrosine kinase, RET/PTC, a genetically rearranged, constitutively active enzyme that plays a pathogenic role in papillary thyroid cancer. We found that XB130 is a RET/PTC substrate that couples RET/PTC signaling to phosphatidylinositol 3-kinase (PI3K) activation through its phosphorylation dependent interaction with the regulatory subunit p85 of PI3K. XB130 plays an important role in PI3K signaling, as downregulation of XB130 in TPC1 papillary thyroid cancer cells, harboring the RET/PTC1 kinase, strongly reduced Akt activity and concomitantly inhibited cell cycle progression and survival in suspension. In the second part we demonstrate that XB130 is a novel Rac- and cytoskeleton-regulated protein that exhibits high affinity to lamellipodial (branched) F-actin and impacts motility and invasiveness of tumor cells. In conclusion, my work characterized a novel adaptor protein and assigned two well-defined pathophysiological functions to it in the context of thyroid cancer cells.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Adaptor proteins play a vital role in the propagation of cellular signals. Although they lack endogenous catalytic activity, they contain a variety of protein binding modules, which enable them to promote specific and efficient interactions with their binding partners. They form integrative platforms for a variety of molecules (e.g. lipids, tyrosine kinases, cytoskeletal and signaling proteins), and thereby link and coordinate key functions such as cell growth, motility and shape determination. Our laboratory has recently cloned a novel, 130 kDa adaptor protein, named XB130, as a structural homolog of actin-filament-associated-protein (AFAP-110), a stress fiber-binding Src substrate. However, the molecular interactions and functions of this novel adaptor remained to be elucidated. To characterize the function of XB130 we asked two general questions: (1) Is XB130 involved in the signal transduction pathways of tyrosine kinases? And (2) Is XB130 capable of regulating the cytoskeleton and/or is it regulated by the cytoskeleton? To address these questions first we investigated the tissue distribution of XB130 and discovered that it is abundantly expressed in thyroid. Therefore we asked whether it is a target of the thyroid-specific tyrosine kinase, RET/PTC, a genetically rearranged, constitutively active enzyme that plays a pathogenic role in papillary thyroid cancer. We found that XB130 is a RET/PTC substrate that couples RET/PTC signaling to phosphatidylinositol 3-kinase (PI3K) activation through its phosphorylation dependent interaction with the regulatory subunit p85 of PI3K. XB130 plays an important role in PI3K signaling, as downregulation of XB130 in TPC1 papillary thyroid cancer cells, harboring the RET/PTC1 kinase, strongly reduced Akt activity and concomitantly inhibited cell cycle progression and survival in suspension. In the second part we demonstrate that XB130 is a novel Rac- and cytoskeleton-regulated protein that exhibits high affinity to lamellipodial (branched) F-actin and impacts motility and invasiveness of tumor cells. In conclusion, my work characterized a novel adaptor protein and assigned two well-defined pathophysiological functions to it in the context of thyroid cancer cells.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.