Molecular Mechanisms in Cell Differentiation and Cell Division

p63 ist eines von drei Mitgliedern der p53 Familie von Transkriptionsfaktoren. Die Transkription des p63 Gens führt zu zwei verschiedenen Isoformen, der Isoform TA-p63 mit einer N-terminalen Transaktivierungsdomäne und der Isoform DeltaN-p63 ohne diese Domäne. Die hier beschriebene Untersuchung der Expression von p63 in Geweben und die Studie von p63-defizienten Mäusen zeigte, dass p63 in der Entwicklung von epithelialen Geweben, des Schädels und der Extremitäten eine wichtige Rolle spielt. Die Bedeutung von p63 in diesen Vorgängen wurde weiterhin verdeutlicht durch das Auftreten von p63 Mutationen in der menschlichen Keimbahn, die zu schweren Defekten in der Entwicklung von Extremitäten und zu epidermalen Fehlbildungen führen. Die genaue Funktion von p63 in der epidermalen Entwicklung ist noch immer stark umstritten. Während einerseits p63 als entscheidender Faktor in der Differenzierung des embryonalen Ektoderms zu epidermalen Geweben beschrieben wird, wird es andererseits als Stammzellfaktor für die Aufrechterhaltung des Teilungsvermögens und der Regeneration von epidermalen Stammzellen beschrieben. Darüber hinaus existieren unterschiedliche Meinungen zu der jeweiligen Bedeutung der TA- und der DeltaN-p63-Isoform in diesen Prozessen. Jüngste Studien haben gefunden, dass TA-p63 eine entscheidende Rolle zum Schutz der weiblichen Keimbahn vor DNS Schädigung spielt. Die vorliegende Arbeit beschreibt die Anfertigung einer TA-p63 defizienten Maus zur Aufklärung der spezifischen Funktion von TA-p63 in der epidermalen Entwicklung und anderen Prozessen in vivo. In der Zellteilung ist es von großer Bedeutung, dass die Aufteilung von Schwesterchromatiden mit hoher Genauigkeit erfolgt. Das Auftreten von Fehlern in diesem Prozess kann zu Zellen mit unterschiedlicher Anzahl von Chromosomen und damit zu Entwicklungsschäden und Tumorerkrankungen führen. Der Spindle Assembly Checkpoint ist ein Schutzmechanismus der Zelle, der Fehler beim Binden der Mikrotubuli an die Kinetochore oder das Fehlen von Spannung zwischen den Kinetochoren bemerkt und die Zelle in der Metaphase aufhält, bis alle Fehler behoben sind. Seit der Entdeckung der ersten Komponenten des Spindle Assembly Checkpoint vor über 15 Jahren, ist ein hochkomplexes Proteinnetzwerk charakterisiert worden, das Schwesterchromatiden-Kohäsion, Kinetochoren und Mikrotubulizytoskelett mit dem Spindle Assembly Checkpoint verbindet. Eines der neuesten Mitglieder dieses Netzwerkes ist die Familie der Shugoshin (Sgo) Proteine. Die folgende Arbeit ist eine detaillierte Charakterisierung des Checkpoint Proteins BubR1, seiner post-translationalen Modifikationen und seiner Interaktionspartner in Interphase und Mitose. Weiterhin wurde die Funktion des mit BubR1 interagierenden Proteins Sgo2 in der Mitose untersucht.p63 is one of three members of the p53 family of transcription factors. Transcription of the p63 gene gives rise to two different N-terminal isoforms, one with (TA-p63) and one without (DeltaN-p63) a transactivation domain. Analysis of p63 protein expression in tissues and of mice deficient for all p63-isoforms revealed a function of p63 in epithelial, craniofacial, and limb development. The significance of p63 in epidermal development is further highlighted by the discovery of p63 germline mutations in severe human syndromes with limb defects and ectodermal dysplasia. Interestingly, the interpretation of p63 function in epidermal development is still controversial. On one hand, p63 is discussed as a commitment factor for the embryonic ectoderms to epidermal lineage, while on the other hand, it is suggested that p63 is a stem cell factor involved in maintenance of proliferative potential and regeneration of epidermal stem cells. Furthermore, there are different opinions about the relative significance of TA- and DeltaN-p63 in the commitment to epidermal lineages and in epidermal differentiation. Recently, studies in mice deficient for more than one p53 family member were conducted to reveal potential cross-regulation between them. Moreover, TA-p63 was found to be implicated in the protection of the female germline by inducing cell death in oocytes upon gamma-irradiation. The work presented here investigates the function of TA-p63 in the commitment of embryonic ectoderm to epidermal lineages and epithelial development to resolve the DeltaN-p63 in this process. Furthermore, mice deficient for TA-p63 mice are described. A high fidelity of chromosome segregation is crucial to ensure correct transmission of genetic material to daughter cells. Errors in this process result in aberrant chromosome numbers and can cause severe developmental defects, miscarriages, and cancer. The spindle assembly checkpoint is a surveillance mechanism that monitors chromosome segregation, detects attachment defects, and delays anaphase onset until errors are corrected. Moreover, passive mechanisms such as kinetochore geometry, architecture, and back-to-back orientation of sister kinetochores further reduce the risk of mis-attachment. Upon satisfaction of the spindle assembly checkpoint, inactivation of Cdk1/cyclin B and cleavage of cohesin leads to chromosome separation and cell cycle progression into anaphase. Since the identification of the first molecular components of the spindle assembly checkpoint over 15 years ago, many proteins were found to be involved in checkpoint signaling. A highly complex protein interaction network is emerging that connects sister chromatid cohesion, kinetochore biology, and microtubule cytoskeleton with the spindle assembly checkpoint. The conserved family of shugoshin proteins are one of the latest additions to this network and present a link between sister chromatid cohesion, checkpoint signaling, and microtubule dynamics. While initial investigations in yeast and drosophilia have been conducted, little is known about shugoshin functions in mammalian cells. The work presented here provides a detailed characterization of one of the key players in checkpoint signaling, BubR1, including its post-translation modifications and its interactions during the cell cycle. Furthermore, the work provides insight into the regulation and evolution of BubR1’s localization and function. Finally, the interaction of BubR1 and Sgo2 is shown to link checkpoint signaling and kinetochore geometry

Yeast Vacuolar HOPS, Regulated by its Kinase, Exploits Affinities for Acidic Lipids and Rab:GTP for Membrane Binding and to Catalyze Tethering and Fusion

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide–sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion

Neurospora WC-1 Recruits SWI/SNF to Remodel Frequency and Initiate a Circadian Cycle

In the negative feedback loop comprising the Neurospora circadian oscillator, the White Collar Complex (WCC) formed from White Collar-1 (WC-1) and White Collar-2 (WC-2) drives transcription of the circadian pacemaker gene frequency (frq). Although FRQ-dependent repression of WCC has been extensively studied, the mechanism by which the WCC initiates a circadian cycle remains elusive. Structure/function analysis of WC-1 eliminated domains previously thought to transactivate frq expression but instead identified amino acids 100–200 as essential for frq circadian expression. A proteomics-based search for coactivators with WCC uncovered the SWI/SNF (SWItch/Sucrose NonFermentable) complex: SWI/SNF interacts with WCC in vivo and in vitro, binds to the Clock box in the frq promoter, and is required both for circadian remodeling of nucleosomes at frq and for rhythmic frq expression; interestingly, SWI/SNF is not required for light-induced frq expression. These data suggest a model in which WC-1 recruits SWI/SNF to remodel and loop chromatin at frq, thereby activating frq expression to initiate the circadian cycle

Insulin Stimulates the Phosphorylation of the Exocyst Protein Sec8 in Adipocytes

The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane

Comparative Analysis of Mutant Huntingtin Binding Partners in Yeast Species.

Huntington\u27s disease is caused by the pathological expansion of a polyglutamine (polyQ) stretch in Huntingtin (Htt), but the molecular mechanisms by which polyQ expansion in Htt causes toxicity in selective neuronal populations remain poorly understood. Interestingly, heterologous expression of expanded polyQ Htt is toxic in Saccharomyces cerevisiae cells, but has no effect in Schizosaccharomyces pombe, a related yeast species possessing very few endogenous polyQ or Q/N-rich proteins. Here, we used a comprehensive and unbiased mass spectrometric approach to identify proteins that bind Htt in a length-dependent manner in both species. Analysis of the expanded polyQ-associated proteins reveals marked enrichment of proteins that are localized to and play functional roles in nucleoli and mitochondria in S. cerevisiae, but not in S. pombe. Moreover, expanded polyQ Htt appears to interact preferentially with endogenous polyQ and Q/N-rich proteins, which are rare in S. pombe, as well as proteins containing coiled-coil motifs in S. cerevisiae. Taken together, these results suggest that polyQ expansion of Htt may cause cellular toxicity in S. cerevisiae by sequestering endogenous polyQ and Q/N-rich proteins, particularly within nucleoli and mitochondria

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment.

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling

Application of RNAi-Induced Gene Expression Profiles for Prognostic Prediction in Breast Cancer

Homologous recombination (HR) is the primary pathway for repairing double-strand DNA breaks implicating in the development of cancer. RNAi-based knockdowns of BRCA1 and RAD51 in this pathway have been performed to investigate the resulting transcriptomic profiles. Here we propose a computational framework to utilize these profiles to calculate a score, named RNA-Interference derived Proliferation Score (RIPS), which reflects cell proliferation ability in individual breast tumors. RIPS is predictive of breast cancer classes, prognosis, genome instability, and neoadjuvant chemosensitivity. This framework directly translates the readout of knockdown experiments into potential clinical applications and generates a robust biomarker in breast cancer

Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation

Akt Regulates TNF? Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNF? production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNF?. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNF? production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation