A novel protein complex has been identified in human cells that has a molecular mass of approximately 450 kDa. It consists of at least eight different subunits including JAB1, the Jun activation-domain binding protein 1, and Trip15, the thyroid hormone receptor-interacting protein 15. The purified complex contains COP9 and COP11 protein homologs and is very similar, if not identical, to the plant COP9 complex involved in light-mediated signal transduction. The isolated JAB1-containing particle has kinase activity that phosphorylates IkappaBalpha, the carboxy terminus of p105, and Ser63 and/or Ser73 of the amino-terminal activation domain of c-Jun. The phosphorylation of c-Jun requires the carboxy terminus of the protein containing the DNA binding and dimerization domains. Three subunits of the new complex--Sgn3, Sgn5/JAB1, and Sgn6--exhibit sequence similarities to regulatory components of the 26S proteasome, which could indicate the existence of common substrate binding sites. Immunofluorescence staining reveals that the new complex shows a subcellular distribution similar to that of the 26S proteasome. The functional relationship of the two particles in regulating transcriptional activity is discussed. Considering the putative role of the complex in signal transduction and its widespread occurrence, we suggest the name JAB1-containing signalosome.

SummaryHelicobacter pylori is one of the most common bacterial pathogens, infecting about 50% of the world population. The presence of a pathogenicity island (PAI) in H. pylori has been associated with gastric disease. We present evidence that the H. pylori protein encoded by the cytotoxin-associated gene A (cagA) is translocated and phosphorylated in infected epithelial cells. Two-dimensional gel electrophoresis (2-DE) of proteins isolated from infected AGS cells revealed H. pylori strain-specific and timedependent tyrosine phosphorylation and dephosphorylation of several 125±135 kDa and 75±80 kDa proteins. Immunoblotting studies, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), cell fractionation and confocal microscopy demonstrated that one of the 125±135 kDa proteins represents the H. pylori CagA protein, which is translocated into the host cell membrane and the cytoplasm. Translocation of CagA was dependent on functional cagA gene and virulence (vir) genes of a type IV secretion apparatus composed of virB4, virB7, virB10, virB11 and virD4 encoded in the cag PAI of H. pylori. Our findings support the view that H. pylori actively translocates virulence determinants, including CagA, which could be involved in the development of a variety of gastric disease.

Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cγ but not Grb2-associated binder 1 or growth factor receptor–bound protein 2. The H. pylori–induced motogenic response is suppressed and blocked by the inhibition of PLCγ and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori–infected epithelial cells suggests that CagA could be involved in tumor progression.

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.

The activation of nuclear factor KB (NF-KB) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-KB/ IKB components following stimulation and show that the final step of NF-KB induction in vivo involves phosphorylation of several members of the NF-KB/IKB protein families. In HeLa cells as well as in B cells, TNF-cx rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-KB precursors and IKBa became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-KB revealed constitutively phosphorylated p65 and IKBBa. Phosphorylation was accompanied by induced processing of the precursors plOO and p105 and by degradation of IKBa. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-KB, as has been shown before for IKBa. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-a selectively activates only specific NF-KB heteromers and that modifications regulate not only IKB molecules but also NF-KB molecules. In fact, cellular NF-KB activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-KB was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-KB translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-a. Thus, phosphorylation appears to be a general mechanism for activation of NF-KB in vivo.

The transcription factor nuclear factor-kappa B (NF-kappaB) is a crucial regulator of many physiological and patho-physiological processes, including control of the adaptive and innate immune responses, inflammation, proliferation, tumorigenesis, and apoptosis. Thus, the tight regulation of NF-kappaB activity within a cell is extremely important. The central mechanism of NF-kappaB regulation is the signal-induced proteolytic degradation of a family of cytoplasmic inhibitors of NF-kappaB, the IkappaBs. However, with the discovery of an IkappaB-independent noncanonical or "alternative" pathway of NF-kappaB activation, the importance of other regulatory mechanisms responsible for the fine-tuning of NF-kappaB became clear. Post-translational modification, especially phosphorylation, of the Rel proteins, of which dimeric NF-kappaB is composed, are such alternative regulatory mechanisms. The best analyzed example is RelA phosphorylation, which takes place at specific amino acids resulting in distinct functional changes of this gene regulatory protein. The interaction of NF-kappaB with other proteins such as glucocorticoid receptors is very important for the regulation of NF-kappaB activity. Recently, exciting new concepts of IkappaB-independent NF-kappaB control like dimer exchange and nucleolar sequestration of RelA have been described, indicating that many aspects of NF-kappaB control are waiting to be discovered.

The COP9 signalosome (CSN) is a conserved protein complex that regulates assembly and activity of cullin-RING ubiquitin ligases (CRLs). Ubiquitin-dependent degradation of the NF-jB inhibitor IjBa preceeds nuclear translocation of NF-jB. For the first time, we show here an inducible interaction of the CSN with IjBa and that the CSN controls IjBa and NF-jB activity. Strikingly, disruption of the CSN by a small interfering RNA-mediated knockdown of single CSN subunits results in a reduced re-accumulation of IjBa and prolonged nuclear translocation of NF-jB in TNFa-stimulated cells. The control of IjBa by the CSN is regulated by deubiquitinylation of IjBa conferred by the CSN-associated deubiquitinylase USP15. Protein expression levels of cullin1 and the CRL substrate adapter b-TrCP are reduced in nonstimulated cells with a disrupted function of the CSN, which might account for an impaired basal turnover of IjBa. We propose that the CSN controls both CRL activity and stability of the CRL substrate IjBa. In consequence, basal and signal-induced CRL-dependent turnover of IjBa is precisely adapted to specific cellular needs.

The NF-kappa B subunits p50 and p65 and the product of the rel proto-oncogene are members of a growing class of transcription factors with a unique DNA-binding and dimerization domain. Nuclear transfer of each of these factors is controlled by cytoplasmic inhibitors, and regulated by specific stimuli. The inhibitors I kappa B-alpha and -beta and pp40 recognize either p65 or the c-rel protein. We show here that the proto-oncogene bcl-3, believed to be involved in certain human B-cell leukaemias, encodes a protein that functions as an I kappa B-like molecule for native NF-kappa B but is specific for the p50 subunit. The ankyrin repeat domain of the bcl-3 product is shown to mediate complex formation with NF-kappa B dimers by contracting the conserved dimerization domain of NF-kappa B.