Charakterisierung von DYRK1A als nicht-kanonischer Modulator des Hedgehog-Signalwegs

Der Hedgehog (HH)-Signalweg spielt eine besondere Rolle während der Embryonalentwicklung. Im adulten Stadium ist er bis auf wenige Ausnahmen inaktiv und eine ektopische Reaktivierung kann zur Entstehung von Krebs führen. 30 % aller Tumorerkrankungen zeigen eine gesteigerte Aktivität des HH-Signalweg...

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Bibliographic Details
Main Author: Schneider, Philipp
Contributors: Lauth, Matthias (Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
Online Access:PDF Full Text
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The Hedgehog (HH) signaling pathway plays a pivotal role during embryogenesis. Except for few exceptions it is inactive in adults and ectopic reactivation can lead to the development of cancer. 30 % of all tumors show increased HH activity. Besides canonical pathway activation by a HH ligand alternative routes leading to increased activation of the GLI-Kruppel family transcription factors GLI1, GLI2 and GLI3 exist. Here dual specificity tyrosine-phosphorylation regulated kinases (DYRKs) play emerging roles. DYRK1B and DYRK2 counteract the HH pathway. In the literature DYRK1A is characterized as a positive factor for GLI1 by direct phosphorylation depending on GLI1’s N terminus bearing activating functions such as an accumulation of GLI1 in the nucleus. The clarification of this mechanism is a worthwhile aim, since protein kinases are ideal targets for highly potent pharmaceuticals. This work confirms the positive effect of transiently overexpressed DYRK1A on GLI1. Serine 102, 104, 130 and 132 were of importance to that suggesting a phosphorylation of these amino residues. The serines 102 and 104 and also 130 and 132 each form together with a proline a general nuclear translocation signal. Besides, this work shows the disassembly of the SUFU-GLI1 complex by DYRK1A depending on its kinase activity but not on the aforementioned serine residues hinting at additional regulatory mechanisms on GLI1 by DYRK1A. Further experiments showed different effects of DYRK1A on HH signaling ranging from the expected stimulatory effect to an inhibition of the signaling pathway. An in vitro kinase screen performed by the group showed the ACTIN binding LIM protein 1 (ABLIM1) as phosphorylation target of DYRK1A and further investigation revealed ABLIM1 (and ABLIM2) and also the downstream effectors MKL1 (megakaryoblastic leukemia 1) and KDM3A (lysine-specific demethylase 3A) as postitve factors for the HH pathway. We hypothesize that DYRK1A phosphorylates ABLIM proteins leading to their release from ACTIN filaments. The consequential increment of the G ACTIN pool leads to an inhibition of MKL1 with the result that it no longer promotes the expression of HH target genes together with KDM3A. This shows a new non-canonical mechanism of HH pathway regulation by DYRK1A that could explain the observed inhibitory role of the kinase. The connection of HH Signaling with the field of MKL1 mediated target gene expression enables a cross-link with a number of cellular processes. The novel insights obtained in this work contribute to the clarification of the complex modulation of the HH pathway und so are an important step for the targeted development of therapeutic approaches concerning tumor diseases resulting from a deregulation of the HH pathway.