Physical and functional characterization of the TBK1/IKKi core complex

Bei einer viralen oder bakteriellen Infektion, ist ein gut funktionierendes Immunsystem überlebenswichtig für den menschlichen Organismus. Ein gutes Verständnis für das Zusammenspiel der Bestandteile des Immunsystems ist daher entscheidend. Verschiedene Rezeptoren des angeborenen Immunsystems erkennen bestimmte molekulare Muster an Krankheitserregern, woraufhin eine große Anzahl von Proteinen an der Signalübertragung beteiligt ist und eine vermehrte Aktivierung antimikrobieller Gene hervorruft. TBK1 (TANK-binding kinase 1) und IKKi (IkappaB kinase-I) sind zwei verwandte Serin/Threonin Kinasen und spielen eine wichtige Rolle bei der Signalübertragung im angeborenen Immunsystem. Nach bakterieller oder viraler Infektion aktivieren die beiden die Transkriptionsfaktoren IRF3/7 und NF-kappaB. Es wurde nachgewiesen, dass TBK1 und IKKi mit drei Bindungsproteinen, TBKBP1, TBKBP2 (TBK binding proteins 1 and 2) und TANK (TRAF family member associated NF-kappaB activator) interagieren (Bouwmeester, Bauch et al. 2004). Der Mechanismus zur Aktivierung von TBK1 und IKKi, und die Rolle der Bindungsproteine in diesem Prozess sind jedoch noch nicht vollkommen geklärt. Das Hauptaugenmerk dieser Studie lag auf der Untersuchung der molekularen Architektur des Komplexes und der Bedeutung der Bindungsproteine bezüglich der Aktivität von TBK1 und IKKi. Um das zu untersuchen führten wir eine systematische TAP Analyse von den am Komplex beteiligten Komponenten, den beiden Kinasen TBK1 und IKKi und den Bindungsproteinen TBKBP1, TBKBP2 und TANK, durch .Obwohl wir die Interaktion zwischen den Bindungsproteinen und TBK1 und IKKi bestätigen konnten, haben wir festgestellt dass die Bindungsproteine selbst nicht aneinander binden. Dieses Ergebnis lässt vermuten dass TBK1 und IKKi wahrscheinlich unabhängige Komplexe mit den verschiedenen Bindungsproteinen formen. Die Verwendung von verschiedenen TBK1 Mutanten und Immunoprezipitationsexperimente haben gezeigt, dass alle drei Bindungsproteine an dieselbe Region in TBK1, die so genannte coled coil 2 Region, binden. Nach Analyse dieser Region konnten wir 2 Aminosäuren (M690 und E696) finden, welche wichtig für die Bindung der drei Bindungsproteine and TBK1/IKKi sind, da Mutationen in diesen Aminosäuren die Bindung von TBK1 an die 3 Bindungsproteine verhindern. Zusätzlich haben wir eine Aminosäure (an Position L693) entdeckt, die speziell die Bindung von TANK an TBK1/IKKi verhindert, ohne die Bindung von TBKBP1 und TBKBP2 an TBK1 zu beeinträchtigen. Das bedeutet dass die drei Bindungsproteine in der gleichen Region von TBK1 binden, allerdings die Interaktion von einzelnen verschiedenen Aminosäuren abhängt. In unseren Überexpressionsexperimenten konnten wir außerdem sehen, dass die Aktivität von TBK1/IKKi nicht unbedingt mit der Bindung an die drei Bindungsproteine korreliert. Diese Daten sind ein Indikator dafür, dass die Bindungsproteine verschiedene Subkomplexe formen, welche sich in ihrer Funktion nicht ersetzen lassen.The proper functioning of the immune system is crucial for a human organism to defend against viral and bacterial infections. Consequently, it is extremely important to understand the complex interplay between molecules of the immune system. Upon infection, several human receptor types recognize conserved molecular patterns that are a feature of pathogens. A large number of proteins then participate in downstream signaling, resulting in expression of antimicrobial genes. TBK1 (TANK-binding kinase 1) and IKKi (IkappaB kinase-I) are two related serine/threonine kinases that play an important role in innate immunity signaling. Upon bacterial and viral infection, TBK1 and IKKi activate the transcription factors IRF3/7 and NF-kappaB. TBK1 and IKKi have been found to interact with three adaptors: TBKBP1, TBKBP2 (TBK binding proteins 1 and 2) and TANK (TRAF family member associated NF-kappaB activator) (Bouwmeester, Bauch et al. 2004). Yet, the mechanism of TBK1 and IKKi activation and, correspondingly, the role of these proteins are still not fully understood. The main focus of this study was to investigate the molecular architecture of this complex and to elucidate the role of the proteins concerning TBK1 activity. In order to achieve this we first performed a systematic TAP analysis of all the different components of the complex: the two kinases (TBK1 and IKKi) and the three adaptor proteins (TBKBP1, TBKBP2 and TANK). Even though we confirmed the interaction of the adaptor proteins to TBK1 and IKKi we didn’t find any of the binding proteins interacting with each other, suggesting that TBK1 and IKKi are most likely forming independent sub-complexes with each of the adaptors. In agreements with this hypothesis, immunoprecipitation experiments suggested that all three adaptor proteins bind to the same region of TBK1 (coiled coil 2). After analyzing the coiled coil 2 structure, we were able to identify single amino acids responsible for the interaction. Amino acids at the position M690 and E696 in TBK1 were important for its binding to all the adaptor proteins because mutation of these residues abolished binding to all of the TBK1 adaptors. On the other hand, mutation of L693 selectively abrogated binding of TANK without affecting binding of TBKBP1 or TBKBP2, indicating that the adaptor proteins bind to the same region but make contacts with different amino acids. Additionally we found that upon overexpression conditions, TBK1 activity was independently of binding to the adaptor proteins. Altogether, these data suggest that each TBK1 adaptor forms a distinct sub-complex that is required for non-redundant functions of TBK1

Novel roles of CDK6 in the hematopoietic system

CDK6 and CDK4 are cyclin-dependent kinases that regulate cell cycle progression. Their role in G1 to S phase transition is well understood and is redundantly performed by both close homologues. Our lab discovered a novel kinase-independent role of CDK6 in regulating transcription that is not shared by CDK4. In this work I focused on discovering the function of CDK6 within the hematopoietic system, particularly in hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) as well as in erythrocytes. Cdk6-deficient mice do not show pronounced abnormalities in the HSC compartment under homeostatic conditions with the exception of increased numbers of HSCs within the most quiescent stem cell compartment (dormant, d-HSC). Quiescent HSCs are required upon hematopoietic stress induced by bone marrow (BM) transplantation or myelosuppression via 5-Fluorouracil. Under these experimental conditions Cdk6-deficient HSCs display a significantly reduced ability to repopulate the hematopoietic system. The impaired stress-induced hematopoiesis is paralleled by the reduced ability to down-regulate Egr1 a prerequisite for d-HSCs to leave quiescence. Transcriptional profiling supported Egr1 as a central regulated gene upon CDK6 loss in HSCs. Our observations also hold true for leukemia. BCR-ABLp210+-infected BM cells from Cdk6-/- mice fail to induce disease in the periphery despite the presence of leukemic stem cells in the BM of recipient mice. Again Egr1 levels are high in Cdk6-/- LSCs. In line, knock-down of Egr1 in Cdk6-/- BCR-ABLp210+ LSKs significantly enhances their potential to form growth-factor independent colonies. These findings define CDK6 as an important regulator of HSC and LSC activation via the regulation of Egr1. In the second part of this thesis a novel role for CDK6 in stabilizing the cytoskeleton of erythrocytes was unraveled. Despite the largely normal hematopoiesis Cdk6-deficient mice are slightly anemic while harboring an increased number of erythroid cells in the BM. No effects of CDK6 loss were found in proliferation or differentiation or within stress erythropoiesis. In contrast, we found that peripheral blood erythrocytes have a decreased life span. CDK6, but not CDK4 protein levels are present in mature blood erythrocytes - predominantly associated with cell membranes. We also detected an impaired F-Actin formation in Cdk6-/- erythroblasts which likely contributes to the decreased stability of the erythrocyte cytoskeleton and hence the reduced life span. Taken together I here describe novel functions for CDK6 within in the hematopoietic compartment. As CDK4/6 kinase inhibitors are entering the clinics my work is of great significance to understand the biology of CDK6 and its functions as transcriptional regulator to improve therapeutic strategies.submitted by Ruth Maria Scheicherhttp://www.bloodjournal.org/content/125/1/90?sso-checked=trueWien, Med. Univ., Diss., 2014OeBB(VLID)301409

Functional Dissection of the TBK1 Molecular Network

TANK-binding kinase 1 (TBK1) and inducible IkB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification–mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I:C) stimulation, we found that TBK1 activation was strictly dependen

CDK6 as a key regulator of hematopoietic and leukemic stem cell activation

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in reg- ulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk62/2 HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more sus- ceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcrip- tional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABLp2101 LSCs. Transplantation with BCR- ABLp2101–infected bone marrow from Cdk62/2 mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk62/2 BCR-ABLp2101 LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Cdk6 contributes to cytoskeletal stability in erythroid cells

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119(+) cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6(-/-) erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6(-/-) erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-) polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitor