The human stress-activated protein kinase-interacting 1 gene encodes JNK-binding proteins

The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. In this study, we have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin 1 alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin 1 polyclonal antibody showed that full-length Sin I and several smaller isoforms are widely expressed. Sin 1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1 alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1 alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK. (c) 2004 Elsevier Inc. All rights reserved.

The JNK are important for development and survival of macrophages

We report in, this study that activation of the JNK by the growth factor, CSF-1 is critical for macrophage development, proliferation, and survival. Inhibition of JNK with two distinct classes of inhibitors, the pharmacological agent SP600125, or the peptide D-JNKI1 resulted in cell cycle inhibition with an arrest at the G(2)/M transition and subsequent apoptosis. JNK inhibition resulted in decreased expression of CSF-1R (c-fins) and Bcl-x(L) mRNA in mature macrophages and repressed CSF-1-dependent differentiation of bone marrow cells to macrophages. Macrophage sensitivity to JNK inhibitors may be linked to phosphorylation of the PU.1 transcription factor. Inhibition of JNK disrupted PUA binding to an element in the c-fins gene promoter and decreased promoter activity. Promoter activity could be restored by overexpression of PUA. A comparison of expression profiles of macrophages with 22 other tissue types showed that genes that signal JNK activation downstream of tyrosine kinase receptors, such as focal adhesion kinase, Nck-interacting kinase, and Rac1 and scaffold proteins are highly expressed in macrophages relative to other tissues. This pattern of expression may underlie the novel role of JNK in macrophages.

Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/ threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK There are now more than 50 proteins shown to be substrates for JNK These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Radioresistant Burkitt's lymphoma cells exhibit defective Mapk signalling

Using a pair of isogenic Burkitt's lymphoma cell lines, one of which is sensitive (BL30A) and the other resistant (BL30K) to apoptosis induced by ionising radiation and exogenous ceramide, we investigated mitogen-activated protein kinase (MAPK) signalling to determine which members of this kinase family are involved in the apoptotic process in these cells. We have previously shown that BL30A cells produce ceramide after irradiation and that this does not occur in BL30K cells (Michael et at. [1997] Cancer Res 57:3600-3605). We show that p38 MAPK is activated transiently in both cells after ionising radiation. On the of her hand, although JNK is rapidly activated in both cells, this activation is only transient in the resistant cells, whereas in the sensitive cells the activation is sustained. Addition of exogenous ceramide resulted in only a transient activation of INK in both cells. Interestingly, ERK activity was decreased in BL30A cells after ceramide treatment, whereas no such decrease occurred in the resistant cells. Treatment of BL30A cells with phorbol ester before irradiation, which blocks the increase in ceramide and apoptosis, also prevents the sustained increase in JNK activity. At the same time, ERK activity is increased. Our results suggest that p38 MAPK is not required for apoptosis signalling in response to ionising radiation in Burkitt's lymphoma cells and that sustained activation of JNK is necessary for apoptosis in these cells. These results also support the hypothesis that a balance between JNK and ERK activity determines cell fate after exposure to ceramide or ionising radiation. In addition, our results suggest different signalling pathways from exogenous ceramide and radiation, supporting the concept of different intracellular pools of active ceramide. Drug Dev. Res. 52:534-541, 2001. (C) 2001 Wiley-Liss, Inc.

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues: highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T-191. and Y-193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T-187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylationactivity. Phosphorylation was confirmed by the use of a phospho-specific T-187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed. (C) 2002 Elsevier Science (USA). All rights reserved.

Subcellular localization determines MAP kinase signal output

The Raf-MEK-ERK MAP kinase cascade transmits signals from activated receptors into the cell to regulate proliferation and differentiation. The cascade is controlled by the Ras GTPase, which recruits Raf from the cytosol to the plasma membrane for activation. In turn, MEK, ERK, and scaffold proteins translocate to the plasma membrane for activation. Here, we examine the input-output properties of the Raf-MEK-ERK MAP kinase module in mammalian cells activated in different cellular contexts. We show that the MAP kinase module operates as a molecular switch in vivo but that the input sensitivity of the module is determined by subcellular location. Signal output from the module is sensitive to low-level input only when it is activated at the plasma membrane. This is because the threshold for activation is low at the plasma membrane, whereas the threshold for activation is high in the cytosol. Thus, the circuit configuration of the module at the plasma membrane generates maximal outputs from low-level analog inputs, allowing cells to process and respond appropriately to physiological stimuli. These results reveal the engineering logic behind the recruitment of elements of the module from the cytosol to the membrane for activation.

Reconstruction and in silico analysis of the MAPK signaling pathways in the human blood fluke, Schistosoma japonicum

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japoncium MAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein beta and alpha-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.