regulation of MAPK signaling pathways through mixed lineage kinase 3

MAPK Kaskaden spielen eine Schlüsselrolle bei der Übertragung und der Umwandlung von extrazellulären Reizen in intrazelluläre Signale und regulieren dadurch unterschiedliche Prozesse wie Differenzierung, Zellproliferation oder Stress Antworten. 'Mixed Lineage Kinasen' (MLKs) bilden eine Familie von Serin/Threonin Proteinkinasen mit mehreren Protein/Protein Interaktionsdomänen (SH3, Cdc42 Rac interactive binding sequence/CRIB, Leuzin Zipper), die am engsten mit der Familie der MAPKK Kinasen verwandt ist. In der voliegenden Arbeit zeigen wir, dass MLK3 Cdc42/Rac induzierte JNK/SAPK Aktivität vermittelt, welches zu einer anschließenden Phosphorylierung und verstärkter transkriptioneller Aktivität des zur AP-1 Familie gehörenden immediate early Gens c-Jun führt. Zusätzlich phosphoryliert MLK3 die dualspezifischen Kinasen MEK1/2 direkt in vitro und in vivo an den für die Aktivierung wichtigen Serinen 217/221. Wohingegen dies nur zu einer ERK Aktivierung in Überexpressionssystemen führte, demonstrieren die Analysen überzeugend, dass endogenes MEK1/2, welches von MLK3 phosphoryliert worden ist, nicht in der Lage ist, diese Aktivität auf das physiologische Substrat ERK1/2 in vitro wie auch in vivo zu übertragen. Wir postulieren daher, dass MLK3 vermittelte MEK1/2 Phosphorylierung die dualspezifischen Kinasen von ERK1/2 Aktivierung entkoppelt. Zusätzlich zeigen wir, dass Überexpression von Wild Typ MLK3 zur morphologischen Transformation von NIH 3T3 Fibroblasten und Wachstum in Soft Agar führt. In Übereinstimmung mit den anderen Analysen ist MEK1/2 stark phosphoryliert jedoch von ERK1/2 Aktivierung in MLK3 transformierten Zellen entkoppelt. Weiterhin kooperiert MLK3 mit aktivierter MEK1 in Foci Bildung, was zeigt, dass MLK3 andere Signalwege als ERK1/2 in der Zelltransformation benutzt. Vielmehr ist in MLK3 transformierten Fibroblasten Wachstumsfaktor-induzierte ERK1/2 Aktivierung und Expression des immediate early Gens junB partiell blockiert. Unsere Analysen zeigen zum ersten Mal für eine MAPKK Kinase, dass sie MAPK Signalwege unterschiedlich reguliert. Während MLK3 auf der einen Seite JNK/SAPK aktiviert, entkoppelt es MEK1/2 induzierte Phosphorylierung von ERK1/2 Aktivierung und blockiert sogar Wachstumsfaktor-induzierte ERK1/2 Ativierung partiell.MAPK cascades play a key role in transducing and converting signals from extracellular stimuli into intracellular signals thereby regulating diverse processes like differentiation, proliferation or stress responses. Mixed lineage kinases (MLKs) form a family of serin/threonin protein kinases with multiple protein/protein interaction domains (SH3, Cdc42 Rac interactive binding sequence/CRIB, leucine zipper), which are most closely related to the MAPKK kinase family. In this work we show that MLK3 mediates Cdc42/Rac induced JNK/SAPK activation resulting in subsequent phosphorylation and enhanced transcriptional activity of the immediate early gene c-jun, which belongs to the family of AP-1 proteins. In addition MLK3 directly phosphorylates the dual specificity kinases MEK1/2 in vitro and in vivo on serines 217/221, which are important for activation. Whereas this only resulted in activation of the ERK1/2 pathway in overexpression systems, the analyses demonstrate convincingly that endogenous MEK1/2 phosphorylated by MLK3 is unable to transmit its activity in vitro and in vivo to its physiological substrat, ERK1/2. Therefore we postulate that MLK3 mediated MEK1/2 phosphorylation uncouples the dual specificity kinases from ERK1/2 activation. Furthermore we show that overexpression of wild type MLK3 leads to morphological transformation of NIH 3T3 fibroblasts and growth in soft agar. Consistent with the other analyses MEK1/2 is highly phosphorylated but uncoupled from ERK1/2 activation in MLK3 transformed cells. In addition MLK3 cooperates with activated MEK1 in foci formation which demonstrates that MLK3 utilizes pathways different from ERK1/2 for cell transformation. Furthermore growth factor induced ERK1/2 activation and induction of the immediate early gene junB is partially blocked in MLK3 transformed fibroblasts. Our data provide evidence for the first time that a MAPKK kinase differentially regulates MAPK pathways. On the one hand MLK3 activates JNK/SAPK but on the other hand it uncouples MEK1/2 phosphorylation from ERK1/2 activation and even blocks growth factor induced ERK1/2 activation partially

Sulfoximines as ATR inhibitors: Analogs of VE-821

The ATM- and Rad3-related (ATR) kinases play a key role in DNA repair processes and thus ATR is an attractive target for cancer therapy. Here we designed and synthesized sulfilimidoyl- and sulfoximidoyl-substituted analogs of the sulfone VE-821, a reported ATR inhibitor. The properties of these analogs have been investigated by calculating physicochemical parameters and studying their potential to specifically inhibit ATR in cells. Prolonged inhibition of ATR by the analogs in a Burkitt lymphoma cell line resulted in enhanced DNA damage and a substantial amount of apoptosis. Together our findings suggest that the sulfilimidoyl- and sulfoximidoyl-substituted analogs are efficient ATR inhibitors

Caspase-dependent cleavage of the mono-ADP-ribosyltransferase ARTD10 interferes with its pro-apoptotic function

ADP-ribosylation is a post-translational modification that regulates various physiological processes, including DNA damage repair, gene transcription and signal transduction. Intracellular ADP-ribosyltransferases (ARTDs or PARPs) modify their substrates either by poly- or mono-ADP-ribosylation. Previously we identified ARTD10 (formerly PARP10) as a mono-ADP-ribosyltransferase, and observed that exogenous ARTD10 but not ARTD10-G888W, a catalytically inactive mutant, interferes with cell proliferation. To expand on this observation, we established cell lines with inducible ARTD10 or ARTD10-G888W. Consistent with our previous findings, induction of the wild-type protein but not the mutant inhibited cell proliferation, primarily by inducing apoptosis. During apoptosis, ARTD10 itself was targeted by caspases. We mapped the major cleavage site at EIAMD406S, a sequence that was preferentially recognized by caspase6. Caspase-dependent cleavage inhibited the pro-apoptotic activity of ARTD10, as ARTD10(1406) and ARTD10(4071025), either alone or together, were unable to induce apoptosis, despite catalytic activity of the latter. Deletion of the Nterminal RNA recognition motif in ARTD10(2571025) also resulted in loss of pro-apoptotic activity. Thus our findings indicate that the RNA recognition motif contributes to the pro-apoptotic effect, together with the catalytic domain. We suggest that these two domains must be physically linked to stimulate apoptosis, possibly targeting ARTD10 through the RNA recognition motif to specific substrates that control cell death. Moreover, we established that knockdown of ARTD10 reduced apoptosis in response to DNA-damaging agents. Together, these findings indicate that ARTD10 is involved in the regulation of apoptosis, and that, once apoptosis is activated, ARTD10 is cleaved as part of negative feedback regulation

HtrA2, taming the oncogenic activities of WT1

Wilms tumor is a pediatric malignancy of the kidneys and is one of the most common solid childhood cancers. The Wilms tumor 1 protein (WT1) is a transcription factor that can either activate or repress genes involved in growth, apoptosis and differentiation. It is frequently mutated or aberrantly expressed in Wilms tumor, where the wild-type protein would normally act as a tumor suppressor. Several studies, however, have found that wild-type WT1 acts as an oncogene in adult tumors, primarily through the inhibition of apoptosis. The expression of WT1 correlates with the aggressiveness of several adult cancers, and its continued expression following treatment is indicative of a poor outcome

Ang-1 and Ang-2 stimulation of macrophages cooperates with TNF to induce monocyte recruitment.

<p>Monocyte migration assays in response to (A) chemotaxis buffer alone (medium) or in combination with Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml), or chemotaxis buffer supplemented with conditioned medium of macrophages polarized in (B) GM-CSF, (C) IFN-γ or (D) IL-10 in the absence (unstimulated) or presence of 24 h stimulation with TNF (10 ng/ml) alone or in combination with Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml). Bars represent the means and SEM of 7–8 independent experiments. Experiments with each polarization condition were performed in parallel, but for ease of analysis are presented as 3 independent graphs. *P<0.05, * P<0.01, between stimulatory conditions, #P<0.05, ##P<0.01, ### P<0.001, compared to conditioned medium. Friedman test was used for statistical analyses.</p

Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype

<div><p>Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 –differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 –differentiated macrophages, expression of multiple chemokines and cytokines, including <i>CXCL3</i>, <i>CXCL5</i>, <i>CXCL8</i>, <i>IL6</i>, and <i>IL12B</i> was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.</p></div