@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{OkgebHofmann2014,
  author    = {Ok [geb. Hofmann], Claudia Barbara},
  title     = {Isoform-spezifische Analyse der PI3-Kinase (Klasse I) im Multiplen Myelom},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108466},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Das Multiple Myelom (MM) ist eine unheilbare Erkrankung, die aus einer klonalen Proliferation maligner Plasmazellen im Knochenmark hervorgeht. Dabei liegt ein komplexes Signalnetzwerk vor, das zum {\"U}berleben und Wachstum der MM-Zellen f{\"u}hrt. Das MM ist durch eine enorme genetische und ph{\"a}notypische Heterogenit{\"a}t gekennzeichnet. Die konstitutive Aktivierung des PI3K/Akt-Signalwegs spielt bei ungef{\"a}hr der H{\"a}lfte der Patienten mit MM eine wichtige Rolle f{\"u}r das {\"U}berleben der MM-Zellen und ist daher ein potentieller therapeutischer Ansatzpunkt. Isoform-spezifische Untersuchungen der katalytischen Untereinheiten der Klasse I-PI3K (p110α, p110β, p110γ, p110δ) sollten zur Erkenntnis f{\"u}hren, welche dieser Isoformen f{\"u}r das MM Zell{\"u}berleben wichtig sind, um spezifischere Behandlungen mit m{\"o}glichst geringen Nebenwirkungen zu erlauben. Daf{\"u}r wurden zun{\"a}chst Isoform-spezifische Knockdown-Experimente mit MM Zelllinien durchgef{\"u}hrt und sowohl deren {\"U}berleben als auch die Aktivierung der nachgeschalteten Komponenten im PI3K Signalweg untersucht. Zur Verifizierung der Ergebnisse wurden sowohl MM Zelllinien als auch Prim{\"a}rzellen mit Isoform-spezifischen PI3K-Inhibitoren behandelt (BYL 719 f{\"u}r p110α, TGX 221 f{\"u}r p110β, CAY10505 f{\"u}r p110γ und CAL 101 f{\"u}r p110δ) und in gleicher Weise untersucht. In beiden Versuchsans{\"a}tzen stellte sich die katalytische Untereinheit p110α als wichtigste Isoform f{\"u}r das {\"U}berleben von MM Zellen mit konstitutiv phosphoryliertem Akt Signal heraus. Weder der Knockdown noch die pharmakologische Inhibition der anderen drei Isoformen (p110β, p110γ, p110δ) f{\"u}hrten in MM-Zelllinien zur Beeintr{\"a}chtigung des Zell{\"u}berlebens. Auch reagierten die Prim{\"a}rzellen von MM Patienten gr{\"o}ßtenteils nicht mit Apoptose auf eine Behandlung mit TGX 221, CAY10505 oder CAL 101. Aufbauend auf der postulierten Bedeutung von p110α, wurde der daf{\"u}r spezifische Inhibitor BYL 719 mit bereits klinisch etablierten Therapeutika in Kombination verwendet, woraus eine im Vergleich zur Einzelbehandlung verst{\"a}rkte Apoptose resultierte. Insgesamt deuten diese Daten darauf hin, dass PI3K/p110α eine therapeutisch nutzbare Zielstruktur zur Behandlung des Multiplen Myeloms darstellt. Daher scheinen weitergehende pr{\"a}-klinische Studien mit p110α Inhibitoren erfolgversprechend.},
  subject      = {Phosphatidylinositolkinase <Phosphatidylinositol-3-Kinase>},
  language  = {de}
}