Transforming growth factor ␤ (TGF-␤)-activated kinase (TAK1) is known for its involvement in TGF-␤ signaling and its ability to activate the p38-mitogen-activated protein kinase (MAPK) pathway. This report shows that TAK1 is also a strong activator of c-Jun Nterminal kinase (JNK). Both the wild-type and a constitutively active mutant of TAK1 stimulated JNK in transient transfection assays. Mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein kinase/ extracellular signal-regulated kinase (SEK1), a dualspecificity kinase that phosphorylates and activates JNK, synergized with TAK1 in activating JNK. Conversely, a dominant-negative (MKK4/SEK1 mutant inhibited TAK1-induced JNK activation. A kinasedefective mutant of TAK1 effectively suppressed hematopoietic progenitor kinase-1 (HPK1)-induced JNK activity but had little effect on germinal center kinase activation of JNK. There are two additional MAPK kinase kinases, MEKK1 and mixed lineage kinase 3 (MLK3), that are also downstream of HPK1 and upstream of MKK4/SEK mutant. However, because the dominant-negative mutants of MEKK1 and MLK3 did not inhibit TAK1-induced JNK activity, we conclude that activation of JNK1 by TAK1 is independent of MEKK1 and MLK3. In addition to TAK1, TGF-␤ also stimulated JNK activity. Taken together, these results identify TAK1 as a regulator in the HPK1 3 TAK1 3 MKK4/ SEK1 3 JNK kinase cascade and indicate the involvement of JNK in the TGF-␤ signaling pathway. Our results also suggest the potential roles of TAK1 not only in the TGF-␤ pathway but also in the other HPK1/ JNK1-mediated pathways.To date, three related mitogen-activated protein kinase (MAPK) 1 cascades that propagate signals from the plasma membrane to the interior of mammalian cells have been identified (1). These three groups of the MAP kinase family include the extracellular signal-regulated kinases (ERKs) (2), p38-MAPKs (3), and the c-Jun N-terminal kinases (JNKs), also known as the stress-activated protein kinases (SAPKs) (4, 5). The ERKs are the central elements in mitogenic signal transduction downstream of Ras (2). p38-MAPKs seem to participate in the signaling pathways of pro-inflammatory cytokines and environmental stresses, such as interleukin-1, tumor necrosis factor ␣, and osmotic shock (1, 3). JNKs/stress-activated protein kinases play a crucial role in the responses stimulated by pro-inflammatory cytokines, environmental stresses, and apoptotic agents (4 -7). Considerable progress has been achieved in identifying the upstream kinases of the JNK cascade. JNK can be phosphorylated and activated by its immediate upstream kinase MAPK kinase 4/SEK1 which itself can be phosphorylated and activated by MEKK1 (8,9). Together with the recent findings from our laboratory that hematopoietic progenitor kinase-1 (HPK1) directly binds to and phosphorylates MEKK1, a simple JNK pathway paradigm can be drawn: HPK1 3 MEKK1 3 MAPK kinase 4/SEK1 3 JNK (10). However, the JNK cascade, like other MAPK cascades, is complicated by the fact that several other kinases su...

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Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene from Caenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins from Entamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed.

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Helicobacter pylori, a Gram-negative, microaerophilic bacterium found in the stomach, is assumed to be associated with carcinogenesis, invasion and metastasis in digestive diseases. Cytotoxin-associated gene A (CagA) is an oncogenic protein of H. pylori that is encoded by a Cag pathogenicity island related to the development of gastric cancer. The epithelial–mesenchymal transition (EMT) is the main biological event in invasion or metastasis of epithelial cells. H. pylori may promote EMT in human gastric cancer cell lines, but the specific mechanisms are still obscure. We explored the underlying molecular mechanism of EMT induced by H. pylori CagA in gastric cancer. In our article, we detected gastric cancer specimens and adjacent non-cancerous specimens by immunohistochemistry and found increased expression of the EMT-related regulatory protein TWIST1 and the mesenchymal marker vimentin in cancer tissues, while programmed cell death factor 4 (PDCD4) and the epithelial marker E-cadherin expression decreased in cancer specimens. These changes were associated with degree of tissue malignancy. In addition, PDCD4 and TWIST1 levels were related. In gastric cancer cells cocultured with CagA expression plasmid, CagA activated TWIST1 and vimentin expression, and inhibited E-cadherin expression by downregulating PDCD4. CagA also promoted mobility of gastric cancer cells by regulating PDCD4. Thus, H. pylori CagA induced EMT in gastric cancer cells, which reveals a new signaling pathway of EMT in gastric cancer cell lines.