No abstract

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an “atypical apoptosis” characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.

Metastasis of primary tumors leads to a very poor prognosis for patients suffering from cancer. Although it is well established that not every tumor will eventually metastasize, it is less clear whether primary tumors acquire genetic alterations in a stochastic process at a late stage, which make them invasive, or whether genetic alterations acquired early in the process of tumor development drive primary tumor growth and determine whether this tumor is going to be metastatic. To address this issue, we tested genes identified in a large-scale comparative genomic hybridization analysis of primary tumor for their ability to confer metastatic properties on a cancer cell. We identified amplification of the ACK1 gene in primary tumors, which correlates with poor prognosis. We further show that overexpression of Ack1 in cancer cell lines can increase the invasive phenotype of these cells both in vitro and in vivo and leads to increased mortality in a mouse model of metastasis. Biochemical studies show that Ack1 is involved in extracellular matrix-induced integrin signaling, ultimately activating signaling processes like the activation of the small GTPase Rac. Taken cancer ͉ metastasis

Interleukin-1 (IL-1) receptor-associated kinases (IRAKs) are central components of Toll/IL-1 receptor (TIR) signaling pathways. In an attempt to discover novel signal transducers in TIR signaling, we identi¢ed human Pellino2 as an interaction partner of IRAK4. Pellino2 interacts with kinaseactive as well as kinase-inactive IRAK1 and IRAK4. Furthermore, Pellino2 is one of the ¢rst substrates identi¢ed for IRAK1 and IRAK4. Functional studies using overexpression or RNAi knock-down of Pellino2 suggest a role of Pellino2 as a sca¡old-ing protein similar to Pellino1. However, unlike Pellino1, Pellino2 does not seem to activate a speci¢c transcription factor, but links TIR signaling to basic cellular processes.

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.Activation of Toll-like receptor (TLR) 4 signaling pathways by microbial products initiates a cascade of events starting at the receptor level and leading eventually to the induction of an array of genes that encode for immune and inflammatory proteins. Ligand binding typically induces receptor dimerization, the recruitment of adaptor molecules, and the activation of a series of kinase cascades. In the case of the lipopolysaccharide (LPS) receptor TLR4 or the lipoprotein receptor TLR2, the adaptor molecules MyD88 (myeloid differentiation factor 88) and Mal (MyD88 adaptor-like) are recruited to activate NF-B and induce proinflammatory cytokines, such as interleukin-1, tumor necrosis factor ␣, and interleukin-6 (1-3). Two other adaptors, TRIF (Toll/interleukin-1 receptor domain-containing inducing interferon-␤) and TRAM (TRIF adaptor molecule), also participate in TLR4 signaling. Recruitment of these proteins leads to the activation of the transcription factor, IRF3 (interferon regulatory factor-3), and the induction of the type I interferons (4 -6).Mal and MyD88 both contain a Toll/interleukin-1 receptor (TIR) domain and function together to optimally transduce signals from TLR2 as well as TLR4. MyD88 differs from Mal in that it possesses an N-terminal death domain responsible for mediating interactions between MyD88 and members of the interleukin-1 receptor-associated kinase (IRAK) family. Four members of this family have been described, and the pathways leading to their activation have recently been dissected (7). In resting cells, IRAK1 can be found in a receptor complex containing MyD88 and Tollip. Following stimulation with LPS, IRAK1 is recruited to the TLR4 receptor complex, where it undergoes phosphorylation by IRAK4 on key threonine residues. This in turn promotes the autophosphorylating activity of IRAK1, its dissociation from the receptor complex, and subsequent interaction with TRAF6 (tumor necrosis factor receptor-associated factor) and a TAK1-TAB1-TAB2 kinase complex leading to the activation of NFB and mitogenactivated protein kinases. IRAK1...

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.Activation of Toll-like receptor (TLR) 4 signaling pathways by microbial products initiates a cascade of events starting at the receptor level and leading eventually to the induction of an array of genes that encode for immune and inflammatory proteins. Ligand binding typically induces receptor dimerization, the recruitment of adaptor molecules, and the activation of a series of kinase cascades. In the case of the lipopolysaccharide (LPS) receptor TLR4 or the lipoprotein receptor TLR2, the adaptor molecules MyD88 (myeloid differentiation factor 88) and Mal (MyD88 adaptor-like) are recruited to activate NF-B and induce proinflammatory cytokines, such as interleukin-1, tumor necrosis factor ␣, and interleukin-6 (1-3). Two other adaptors, TRIF (Toll/interleukin-1 receptor domain-containing inducing interferon-␤) and TRAM (TRIF adaptor molecule), also participate in TLR4 signaling. Recruitment of these proteins leads to the activation of the transcription factor, IRF3 (interferon regulatory factor-3), and the induction of the type I interferons (4 -6).Mal and MyD88 both contain a Toll/interleukin-1 receptor (TIR) domain and function together to optimally transduce signals from TLR2 as well as TLR4. MyD88 differs from Mal in that it possesses an N-terminal death domain responsible for mediating interactions between MyD88 and members of the interleukin-1 receptor-associated kinase (IRAK) family. Four members of this family have been described, and the pathways leading to their activation have recently been dissected (7). In resting cells, IRAK1 can be found in a receptor complex containing MyD88 and Tollip. Following stimulation with LPS, IRAK1 is recruited to the TLR4 receptor complex, where it undergoes phosphorylation by IRAK4 on key threonine residues. This in turn promotes the autophosphorylating activity of IRAK1, its dissociation from the receptor complex, and subsequent interaction with TRAF6 (tumor necrosis factor receptor-associated factor) and a TAK1-TAB1-TAB2 kinase complex leading to the activation of NFB and mitogenactivated protein kinases. IRAK1...

Ceramide has been implicated in the activation of stress-activated protein kinases\c-Jun N-terminal kinases (SAPK\JNK). Binding of tumour necrosis factor (TNF) to its 55 kDa receptor (TR55) leads to the generation of ceramide through activation of either acid or neutral sphingomyelinase (A\N-SMase) as well as to potent activation of SAPK\JNK. We have examined a putative role of both N-and A-SMase in the TR55-dependent activation of SAPK\JNK. The analysis of TR55 deletion mutants expressed in 70Z\3 pre-B cells revealed that activation of SAPK\JNK occurs independently of N-SMase. Although both SAPK\JNK and A-SMase are activated by the death domain of TR55, pharmacological prevention of the TR55-dependent activation

The Vibrio cholerae transcriptional regulator ToxR is anchored in the cytoplasmic membrane by a single transmembrane segment, its C-terminal domain facing the periplasm. Most of its N-terminal cytoplasmic domain shares sequence similarity with the winged helix-turn-helix (wHTH) motif of OmpR-like transcriptional regulators. In the heterologous host Escherichia coli ToxR activates transcription at the V.cholerae ctx promoter in a dimerization-dependent manner, which has led to its employment as a genetic indicator for protein-protein interactions. However, although offering a broader potential application range than other prokaryotic two-hybrid systems described to date, ToxR has so far only been used to study interactions between heterologous transmembrane segments or to monitor homodimerization of C-terminal fusion partners in the periplasm and the cytoplasm of E.coli. Here we show that the ToxR-system also allows the detection of heterodimerization in both cellular compartments of E.coli. In addition, to better understand ToxR's mode of action at ctx in E.coli, we have investigated the minimal requirements for its function as a transcriptional activator. We show that the wHTH motif of ToxR's Nterminal domain constitutes the minimal structural element required to activate transcription at ctx in E.coli when fused to a dimerizing protein module.