@phdthesis{Sibilski2014,
  author    = {Sibilski, Claudia},
  title     = {Identification and characterization of the novel mKSR1 phosphorylation site Tyr728 and its role in MAPK signaling},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114672},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.},
  subject      = {MAP-Kinase},
  language  = {en}
}
@phdthesis{Gromova2007,
  author    = {Gromova, Kira V.},
  title     = {Visualization of the Smad direct signaling response to Bone Morphogenetic Protein 4 activation with FRET-based biosensors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-25855},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2007},
  abstract  = {The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor - Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs.},
  subject      = {FRET},
  language  = {en}
}
@phdthesis{Spiliotis2006,
  author    = {Spiliotis, Markus},
  title     = {Untersuchungen zur in vitro Kultivierung und Charakterisierung von MAP-Kinase-Kaskade-Komponenten des Fuchsbandwurmes Echinococcus multilocularis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19385},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {Es wird angenommen, dass die invasiven Stadien parasit{\"a}rer Helminthen zur Organfindung und zur Weiterentwicklung auf die Sensierung spezifischer Wirts-Signale angewiesen sind, wobei die molekulare Natur dieser Signale bislang weitgehend ungekl{\"a}rt ist. Vorangegangene Untersuchungen am Fuchsbandwurm Echinococcus multilocularis, dem Erreger der alveol{\"a}ren Echinokokkose, hatten bereits ergeben, dass dessen Metacestoden-Larvenstadium zur Weiterentwicklung kleine, l{\"o}sliche Wirtsmolek{\"u}le ben{\"o}tigt. In der vorliegenden Arbeit wurde erstmals ein axenisches (Wirtszell-freies) Kultursystem f{\"u}r das Metacestoden-Stadium entwickelt, mittels dessen sich diese Fragestellungen in vitro angehen lassen. Mit Hilfe dieses Kultursystems konnte in der vorliegenden Arbeit gezeigt werden, dass die drei Wirts-Hormone/Zytokine, Insulin, epidermal growth factor (EGF) und bone morphogeneic protein 2 (BMP2), einen Einfluss auf die Proliferation und die Differenzierung von E. multilocularis haben. W{\"a}hrend f{\"u}r Insulin und EGF Wachstums-stimulierende Effekte gezeigt werden konnten, f{\"o}rderte BMP2 die Differenzierung des Metacestoden zum n{\"a}chsten Larvenstadium, dem Protoscolex. In Modellorganismen wie S{\"a}ugern, Drosophila und Caenorhabditis elegans verlaufen die durch Insulin- und EGF-{\"a}hnlichen Zytokine induzierten Signalmechanismen {\"u}ber die sogenannte mitogen activated protein (MAP)-Kinase-Kaskade. Um zu untersuchen, ob die externe Zugabe von Wirts-Insulin bzw. -EGF in einer Stimulierung der MAPK-Kaskade des Parasiten f{\"u}hrt, wurden in dieser Arbeit zun{\"a}chst die Komponenten dieses Signalweges bei E. multilocularis auf molekulargenetischer und biochemischer Ebene charakterisiert. Die Arbeiten umfassten Studien zu kleinen GTPasen des Parasiten (EmRas, EmRap1, EmRap2, EmRal), zu einem Orthologen der Kinase Raf (EmRaf), sowie Orthologen der Kinasen MEK (EmMKK) und ERK (EmERK). Es konnte gezeigt werden, dass diese Faktoren in E. multilocularis Teil einer MAP-Kinase-Kaskade sind. Zudem wurde nachgewiesen, dass diese Faktoren stromabw{\"a}rts eines EGF-Rezeptor-Orthologen (EmER) des Parasiten fungieren, welches ebenfalls in der vorliegenden Arbeit analysiert wurde. Damit wurden die Voraussetzungen geschaffen, den Einfluss exogen zugegebenen Insulins bzw. EGFs auf die Aktivierung der MAP-Kinase-Kaskade im Parasiten zu untersuchen. Erste Analysen zeigten bereits, dass die zentrale Komponente dieser Kaskade, EmERK, durch die genannten Wirts-Zytokine aktiviert wird. Dies legt nahe, dass Wirt-Parasit-Kommunikationsmechanismen {\"u}ber evolutionsgeschichtlich konservierte Signalsysteme eine wichtige Rolle im Infektionsgeschehen der alveol{\"a}ren Echinokokkose spielen. Aufbauend auf dem axenischen Kultursystem ist es in dieser Arbeit auch erstmals gelungen, Prim{\"a}rzellkulturen f{\"u}r E. multilocularis anzulegen und die Parasitenzellen zur in vitro Neubildung von Metacestoden-Vesikeln anzuregen. Erste Experimente zur genetischen Manipulation dieser Prim{\"a}rzellen konnten erfolgreich durchgef{\"u}hrt werden. Aufbauend auf der hier vorgestellten Methodik sollte es in k{\"u}nftigen Untersuchungen m{\"o}glich sein, stabil transfizierte Echinococcus-Zellen zu generieren und diese zur Herstellung vollst{\"a}ndig transgener Parasiten-Stadien zu nutzen. Dies w{\"u}rde die zur Untersuchung der E. multilocularis-Entwicklung und der Wirt-Parasit-Interaktionsmechanismen bei einer Infektion zur Verf{\"u}gung stehenden Methoden entscheidend erweitern und k{\"o}nnte u.a. zur weiteren biochemischen Analyse der in dieser Arbeit dargestellten Signalmechanismen des Parasiten herangezogen werden.},
  subject      = {Fuchsbandwurm},
  language  = {de}
}