The c-Jun NH2-terminal kinases (JNK) are evolutionarily conserved serine/threonine protein kinases that are activated by proinflammatory cytokines, environmental stress, and genotoxic agents. These kinases play key regulatory roles within a cell by coordinating signals from the cell surface to nuclear transcription factors. JNK phosphorylates the amino terminal domain of all three Jun transcription factors (JunB, c-Jun and JunD) all members of the AP-1 family. The activated transcription factors modulate gene expression to generate appropriate biological responses, including cell migration, proliferation, differentiation and cell death. The role of the JNK signaling pathway in cell death/apoptosis is controversial, both pro-apoptotic and pro-survival roles have been attributed to JNK. The mechanism that enables the JNK signaling pathway to mediate both apoptosis and survival is unclear. The aim of this study is to examine the role of TNF-stimulated JNK activation on cell survival. The proinflammatory cytokine TNF, is known to activate JNK and induce apoptosis. To test whether the JNK signaling pathway contributes to TNF-induced apoptosis, the response of wild type and Jnk1-/- Jnk2-/- (JNK deficient fibroblasts) fibroblasts to TNF was examined. JNK deficient fibroblasts are more sensitive to TNF-induced apoptosis than wild-type fibroblasts. The TNF-sensitivity cannot be attributed to altered expression of TNF receptors or defects in the NF-кB or AKT pathways, known anti-apoptotic signal transduction pathways. (In fact, TNF stimulated NF-кB activation provides a major mechanism to account for survival in both wild-type and JNK deficient cells.) However this increased TNF-sensitivity can be attributed to JNK deficiency. Apoptosis is suppressed in JNK deficient cells when transduced with JNK1 retrovirus. These data implicate the JNK signaling pathway in cell survival. The AP-1 family of transcription factors is a target of the JNK signal transduction pathway. In addition JNK is required for the normal expression of the AP-1 family member, JunD. Previous studies have indicated that JunD can mediate survival. Interestingly, JNK deficient and JunD null cells display similar phenotypes: premature senescence and increased sensitivity to TNF induced apoptosis. In fact, the TNF-sensitivity is also suppressed in JNK deficient fibroblasts transduced with JunD retrovirus. Although JunD can replace the survival signaling role of JNK, phosphorylation of JunD is essential to inhibit TNF induced apoptosis. JNK deficient cells transduced with phosphomutant JunD retrovirus maintain TNF-sensitivity. Activated transcription factors modulate gene expression. It is most likely that JunD functions by regulating the expression of key molecules that act to inhibit TNF-stimulated apoptosis. Microarray analysis comparing wild-type with JNK deficient fibroblasts revealed that the expression of the survival gene, cIAP-2, was induced by TNF in only wild-type fibroblasts. Furthermore, protein expression of cIAP-2 was induced by TNF in only wild-type fibroblasts. Analysis of the cIAP-2 promoter revealed two critical NF-кB binding sites and one AP-1 binding site. Luciferase reporter assays indicated key roles for both NF-кB and the AP-1 component, JunD in TNF-induced cIAP-2 gene expression. These experiments establish that the JNK/JunD pathway collaborates with NF-кB pathway to increase the expression of the anti-apoptotic protein cIAP-2 in TNF treated cells. Without this collaboration, the JNK pathway mediates apoptosis. The integration of JNK signaling with other signaling pathways represents a mechanism to account for the dual ability of the JNK pathway to mediate either survival or apoptosis. The dynamic coordination of signals within and between pathways is critical. The future challenge will be to fit the details of individual signaling pathways into the context of signaling networks.

A monoclonal antibody, mAb 203.37, raised against purified nuclear matrix proteins identified a single ~270 kDa protein that localized to the nuclear envelope. Double-label immunofluorescent microscopy using differential permeabilization protocols showed that this protein was present exclusively on the nucleoplasmic side of the nuclear envelope and that it co-localized with components of the nuclear pore complex. The nucleotide sequence of clones isolated using mAb 203.37 identified this protein as Tpr, a protein previously shown to be involved in oncogenic fusions with a number of protein kinases. Sequence analysis showed Tpr to be a 2348 amino acid protein with a predicted molecular weight of 265 kDa protein and a bipartite structure consisting of an ~1600 amino acid N-terminal domain that is almost entirely an α-helical coiled-coil followed by a highly acidic non-coiled carboxy-terminus. Ectopic expression of epitope-tagged Tpr constructs revealed two functional domains for Tpr: a nuclear pore complex binding domain and a nuclear localization sequence. The amino-terminus of Tpr, the portion of the protein shown to activate protein kinase oncogenes, did not localize to the nuclear pore complex indicating that the transforming activity of Tpr-protein kinase chimeras did not involve interactions with the nuclear pore complex. Ectopic expression of Tpr and a number of Tpr constructs resulted in the accumulation of poly (A)+ RNA in the nuclear interior but did not effect the import of a reporter protein into the nucleus indicating a role for Tpr in the export of mRNA from the nucleus.

Tyrosine phosphorylation of proteins by protein tyrosine kinases is an important step in initiating mitogenic signal transduction pathways. The receptor tyrosine kinases represent a class of protein kinases that employ phosphorylation cascades to transmit a signal generated at the cell surface. The AP-1 transcription factor is a common target of receptor tyrosine kinase activation, transformation by Ras-like proteins and activation of the MAP kinase pathway. The AP-1 complex contains a dimer of Jun proteins or a heterodimer of Jun and Fos or other bZip proteins. The transcriptional activation of Jun is enhanced by phosphorylation on residues Ser-63 and Ser-73. Therefore, identifying the regulatory proteins kinases of Jun would be an important link in signaling from the upstream cell surface events to downstream events, such as gene expression. The JNK1 protein kinase was identified and phosphorylates c-Jun at these sites. The JNK1 protein is a member of the JNK group of protein kinases, which are activated in response to UV treatment. JNK1 is the 46 kDa isoform, and the isolation of the 55 kDa isoform is described in this thesis. Furthermore, a role for JNK was established in Drosophila. Drosphila JNK (DJNK) is essential for the process of dorsal closure. The JNK protein kinases are involved in cytokine signaling, response to environmental stress and development.

Amplification and/or overexpression of the Mdm2 oncogene occurs in many human cancers. Mdm2 promotes cellular proliferation, interferes with apoptosis, and induces tumor formation through the negative regulation of the p53 tumor suppressor. More than thirty percent of human tumors overexpressing Mdm2 also present with alternatively spliced Mdm2 isoforms that cannot directly bind p53. The presence of Mdm2 isoforms in tumors correlates with a higher tumor grade and a poorer prognosis for the patient. To investigate the function of Mdm2 isoforms in tumorigenesis, we have isolated a number of Mdm2 splice forms from tumors obtained from Mdm2-transgenic mice and find that the most frequently observed splice form in human tumors, Mdm2-b, is conserved in mice. Although the Mdm2-b protein is incapable of binding to p53 and is unable to localize to the nucleus, we demonstrate that Mdm2-b promotes cell growth in NIH3T3 cells, Rb-deficient, p19-deficient, and p53-deficient primary cells. We also show that Mdm2-b inhibits apoptosis in response to serum withdrawal and restimulation, doxorubicin treatment, and TNF-alpha administration. Mdm2-b induces foci formation in vitro and directly contributes to tumor formation in GFAP-Mdm2 transgenic mice. We propose that Mdm2-b promotes tumor growth by upregulating RelA (P65) protein levels and activity in a p53-independent manner. To better understand additional functions of Mdm2 that are p53-dependent, we have generated an Mdm2 conditional mouse model. Using primary mouse embryonic fibroblasts derived from Mdm2 conditional mice, we demonstrate that p21 is required for p53-dependent apoptosis initiated by Mdm2 loss. In support of this observation, we also note that p21-loss partially rescues embryonic lethality of Mdm2 null mice. We further show that p21-loss partially rescues the embryonic lethality caused by the loss of the Mdm2 family member, Mdm4. We address the possibility that Mdm2 and Mdm4 may play redundant roles during embryonic development and find that Mdm2 overexpression fully rescues the embryonic lethality resulting from Mdm4 loss. Our findings demonstrate that both Mdm2 and Mdm4 play critical roles in modulation of the p53 tumor suppressor pathway and that their deregulation can result in tumor formation through both p53-dependent and independent pathways.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Stress-induced JNK activity has been implicated in apoptosis. Gene disruption studies have established that JNK signaling is required for some forms of apoptosis. However, it was not clear whether and how JNK was able to deliver an apoptotic signal, because JNK and its regulated-downstream transcriptional factors control a variety of gene activities and multiple biological functions. I have studied this question by using constitutively activated JNK that is independent of upstream signaling. The results indicate that activated JNK is sufficient to deliver an apoptotic signal that causes cytochrome c release from mitochondria. Significantly, this apoptotic signal requires pro-apoptotic Bc12 proteins of Bax and Bak to mediate the downstream apoptotic program. This part of work established the apoptotic activity of JNK signal transduction and the key downstream components of JNK-stimulated apoptotic signal. Two pathways are known to mediate apoptosis in response to apoptotic stimulations: death receptor pathway and mitochondrial pathway. It has been established that JNK is required for the apoptosis mediated by mitochondria in response to ultraviolet irradiation and some genetic stress. However, the mechanisms are not fully understood. It is well known that Bax and Bak are indispensable downstream components leading to apoptotic mitochondrial changes and that other Bc12 family members can regulate the relative apoptotic activity of Bax and Bak. In conjunction with the first part of the research, I have investigated the hypothesis that JNK-mediated regulation of BH3-only Bc12 members contributes to its apoptotic activity. These results indicate that JNK-mediated phosphorylation of Bim and Bmf promotes the release of these proapoptotic BH3-only proteins from their sequestration and these factors become free to initiate apoptosis. This part of work established one mechanism of activated JNK-stimulated apoptosis. This mechanism may contribute to the phenomenon that Jnk1-/-Jnk2-/- fibroblasts are resistant to ultraviolet irradiation-induced apoptosis.

Many polypeptide growth factors act by binding to cell surface receptors that have intrinsic tyrosine kinase activity. Binding of these growth factors to their cognate receptors results in the initiation of mitogenic signals which then get transduced to the interior of the cell. A critical target for extracellular signals is the nucleus. A plethora of recent evidence indicates that extracellular signals can affect nuclear gene expression by modulating transcription factor activity. In this study, I have determined that the transactivation domain of c-Myc (protein product of the c-myc proto-oncogene) is a direct target of mitogen-activated signaling pathways involving protein kinases. Further, my study demonstrates that transactivation of gene expression by c-Myc is regulated as a function of the cell cycle. c-Myc is a sequence-specific DNA binding protein that forms leucine zipper complexes and can act as a transcription factor. Although, significant progress has been made in understanding the cellular properties of c-Myc, the precise molecular mechanism of c-Myc function in oncogenesis and in normal cell growth is not known. I have focused my attention on the property of c-Myc to function as a sequence-specific transcription factor. In my studies, I have employed a fusion protein strategy, where the transactivation domain of the transcription factor c-Myc is fused to the DNA binding domain and nuclear localization signal of the yeast transcription factor GAL4. This fusion protein was expressed together with a plasmid consisting of specific GAL4 binding sites cloned upstream of a minimal E1b promoter and a reporter gene. The activity of the c-Myc transactivation domain was measured as reporter gene activity in cell extracts. This experimental approach enabled me to directly monitor the activity of the c-Myc transactivation domain. Results listed in Chapter II demonstrate that the transactivation domain of c-Myc at Ser-62 is a target of regulation by mitogen-stimulated signaling pathways. Furthermore, I have determined that a mitogen activated protein kinase, p41mapk, can phosphorylate the c-Myc transactivation domain at Ser-62. Phosphorylation at this site results in a marked increase in transactivation of gene expression. A point mutation at the MAP kinase phosphorylation site (Ser-62) causes a decrease in transactivation. c-Myc expression is altered in many types of cancer cells, strongly implicating c-myc as a critical gene in cell growth control. The molecular mechanisms by which c-Myc regulates cellular proliferation are not understood. For instance, it is not clear where in the cell cycle c-Myc functions and what regulates its activity. In exponentially growing cells, the expression levels of c-Myc remain unchanged as the cells progress through the cell cycle. The function of c-Myc may therefore be regulated by a mechanism involving a post-translational modification, such as phosphorylation. Results described in chapter IV demonstrate that the level of c-Myc mediated transactivation oscillates as cells progress through the cell cycle and was greatly increased during the S to G2/M transition. Furthermore, mutation of the phosphorylation site Ser-62 in the c-Myc transactivation domain diminishes this effect, suggesting a functional role for this phosphorylation site in the cell cycle-specific regulation of c-Myc activity. Taken together, my dissertation study reveals a molecular mechanism for the regulation of nuclear gene expression in response to mitogenic stimuli.

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Background: In addition to patient-related systemic factors directing the immune response, the pathomechanisms of appendicitis (AP) might also include insufficient drainage leading to inflammation caused by decreased peristalsis. Genetic predisposition accounts for 30%–50% of AP. M. Hirschsprung (HSCR), also characterized by disturbed peristalsis, is associated with variants in the RET proto-oncogene. We thus hypothesized that RET variants contribute to the etiology of AP. Methods: DNA from paraffin-embedded appendices and clinical data of 264 children were analyzed for the RET c.135A>G variant (rs1800858, NC_000010.11:g.43100520A>G). In 46 patients with gangrenous or perforated AP (GAP), peripheral blood DNA was used for RET sequencing. Results: Germline mutations were found in 13% of GAP, whereas no RET mutations were found in controls besides the benign variant p.Tyr791Phe (NC_000010.11:g.43118460A>T). In GAP, the polymorphic G-allele in rs2435352 (NC_000010.11:g.43105241A>G) in intron 4 was underrepresented (p = 0.0317). Conclusion: Our results suggest an impact of the RET proto-oncogene in the etiology of AP. Mutations were similar to patients with HSCR but no clinical features of HSCR were observed. The pathological phenotypes in both populations might thus represent a multigenic etiology including RET germline mutations with phenotypic heterogeneity and incomplete penetrance.