Identification of a member of the MAPKKK family as a potential mediator of TGF-beta signal transduction.

The mitogen-activated protein kinase (MAPK) pathway is a conserved eukaryotic signaling module that converts receptor signals into various outputs. MAPK is activated through phosphorylation by MAPK kinase (MAPKK), which is first activated by MAPKK kinase (MAPKKK). A genetic selection based on a MAPK pathway in yeast was used to identify a mouse protein kinase (TAK1) distinct from other members of the MAPKKK family. TAK1 was shown to participate in regulation of transcription by transforming growth factor-beta (TGF-beta). Furthermore, kinase activity of TAK1 was stimulated in response to TGF-beta and bone morphogenetic protein. These results suggest that TAK1 functions as a mediator in the signaling pathway of TGF-beta superfamily members.

TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Possible transsynaptic cholinergic neuromodulation by ATP released from ileal longitudinal muscles of guinea pigs.

The effects of alpha, beta-methylene ATP (alpha, beta-mATP) and beta, gamma-methylene ATP (beta, gamma-mATP) on endogenous acetylcholine (ACh) release evoked by electrical nerve stimulation were evaluated in guinea-pig ileal longitudinal muscles. Release of ACh was measured with an HPLC-electrochemical detector system and release of ATP by luciferin-luciferase assay. Electrically evoked endogenous ACh release was reduced by both alpha, beta-mATP and beta, gamma-mATP at concentrations of 3 and 30 microM. The inhibitory effect of alpha, beta-mATP (30 microM) on ACh release was not detectable in the presence of theophylline (100 microM), a P1-purinoceptor antagonist, that itself enhanced ATP release. When exogenous ATP (0.1 microM) was added to the bath in which the ileal segment was suspended, it was rapidly metabolized, presumably by ecto-ATPase, and disappeared from the medium within 15 min. At 30 microM, alpha, beta-mATP induced ATP release in a suramin-sensitive but Ca(2+)- and atropine-insensitive manner, suggesting P2-receptor-mediated release of ATP from the smooth muscle. We conclude from these findings that alpha, beta-mATP and, probably, also beta, gamma-mATP, do not reduce ACh release by direct stimulation of presynaptic P1-purinoceptors, and that endogenous ATP released postjunctionally by these ATP analogs is decomposed metabolically to adenosine in the synapse and this adenosine triggers P1-purinoceptor sensitive neuromodulation of cholinergic transmission.

A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator.

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.

Roles of Meltrin beta /ADAM19 in the processing of neuregulin.

Meltrin beta/ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin beta during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin beta participates in the proteolytic processing of membrane-anchored NRGs. When NRG-beta1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin beta, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-beta1. Furthermore, expression of protease-deficient mutants of meltrin beta exerted dominant negative effects on the basal processing of NRG-beta1. These results indicate that meltrin beta participates in the processing of NRG-beta1. Since meltrin beta affected the processing of NRG-beta4 but not that of NRG-alpha2, meltrin beta was considered to have a preference for beta-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin beta participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.

Antitumor activity of a benzaldehyde derivative.

Benzaldehyde, in the form of 4,6-benzylidene-alpha-D-glucose (BG), was given iv at a daily dose of 720-1800 mg/m2 to 65 patients with inoperable carcinoma in the advanced stages. The overall objective response rate was 55%; seven patients achieved complete response, 29 achieved partial response, 24 remained stable, and five showed progressive disease. Response was seen in various cell types. Prolongation of survival was apparent for the patients. Toxic reactions were not observed during long-term injection with BG.

Initiation of Xenopus oocyte maturation by activation of the mitogen-activated protein kinase cascade.

Mitogen-activated protein kinase (MAPK) and MAPK kinase (MAPKK) are activated during Xenopus oocyte maturation concomitant with the activation of maturation promoting factor (MPF). We reported previously that an anti-MAPKK neutralizing antibody inhibited progesterone- or Mos- induced initiation of oocyte maturation. Here, we show that the injection of CL100 (also called MAPK phosphatase-1) into immature oocytes inhibited progesterone-induced oocyte maturation as well as MAPK activation and that injection of mRNA encoding a constitutively active MAPKK induced activation of histone H1 kinase and germinal vesicle breakdown in the absence of progesterone. Injection of recombinant STE11 protein (a yeast MAPKK kinase) also induced initiation of oocyte maturation. These data support the idea that the MAPKK/MAPK cascade plays an important role in oocyte maturation. Interestingly, injection of the active MAPKK mRNA or the STE11 protein resulted in induction and accumulation of Mos protein. Furthermore, in the presence of cycloheximide, the STE11-induced activation of MPF as well as the induction and accumulation of Mos was blocked, and the activation of MAPK was greatly reduced. The increase in Mos protein and the activation of MAPK by injecting cyclin A protein into immature oocytes were both blocked also by cycloheximide treatment. These results are consistent with an idea that there may exist a positive feedback loop consisting of Mos, the MAPKK/MAPK cascade, and MPF, which may be important for the initiation of oocyte maturation induced by progesterone.

Involvement of dihydropyridine-sensitive Ca2+ channels in adenosine-evoked inhibition of acetylcholine release from guinea pig ileal preparation.

The effects of adenosine and nifedipine on endogenous acetylcholine (ACh) release evoked by electrical stimulation from guinea pig ileal longitudinal muscle preparations exposed to physostigmine were evaluated using an HPLC with electrochemical detection (ECD) system. Resting ACh release, which was sensitive to tetrodotoxin (0.3 microM), was enhanced by Bay K 8644 (0.5 microM; a Ca2+ antagonist) or 4-aminopyridine (30 microM; a K+ channel blocker) but not by theophylline (100 microM; a P1 purinoceptor antagonist) or atropine (0.3 microM). The enhancement of the resting ACh release by Bay K 8644 was virtually unaffected by atropine. Electrically evoked ACh release was enhanced by around two- to fourfold in the presence of theophylline, atropine, Bay K 8644, 4-aminopyridine, or atropine. On the other hand, the evoked ACh release was reduced by adenosine (10-30 microM), nifedipine (0.1-0.3 microM; a dihydropyridine Ca2+ channel antagonist), or bethanechol (1-3 microM) in a concentration-related fashion. The reduction induced by adenosine or nifedipine was almost abolished by either theophylline or Bay K 8644, whereas that induced by bethanechol was virtually unaffected by these drugs. The inhibition by adenosine of ACh release was not influenced in the presence of 4-aminopyridine or atropine. However, this inhibition by adenosine was considerably enhanced by halving the Ca2+ concentration in the Krebs solution and was diminished by doubling the Ca2+ concentration. These findings suggest that adenosine produces a cholinergic neuromodulation presumably via modifying dihydropyridine-sensitive Ca2+ channel activities in the cholinergic neurons, and thus L-type Ca2+ channels may exist on the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5.

BACKGROUND: Runx2, formerly called PEBP2alphaA or Cbfa1, is a transcription factor whose deletion causes a complete lack of ossification. It directly regulates the expression of osteoblast-specific genes through the osteoblast-specific cis-acting element found in the promoter region of these genes. RESULTS: In this study, we have found conditions in which induction of the expression of Runx2 is not accompanied by expression of an osteoblast-specific gene, osteocalcin in C2C12 cells. This finding suggests the existence of a repressor of the activity of Runx2. We have then found that the homeobox protein Msx2 is able to repress the transcription activity of Runx2 by interacting with it. Furthermore, our results have shown that the other homeobox protein Dlx5 has an activity which interferes with both abilities of Msx2 to interact with Runx2 and repress its transcription activity. It has previously been shown that a missense mutation of Msx2 (P148H) causes Boston-type craniosynostosis in humans. Interestingly, while this mutant form of Msx2 was able to bind to Runx2 and repress its activity, these abilities of Msx2 (P148H) were not subject to regulation by Dlx5. CONCLUSION: These results suggest that regulation of the activity of Runx2 by Msx2 and Dlx5 plays an important role in the mammalian skull development.

TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase.

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes. It has recently been reported that ceramide activates stress-activated protein kinase (SAPK, also known as c-Jun NH2-terminal kinase JNK), a subfamily member of mitogen-activated protein kinase superfamily molecules and that the ceramide/SAPK/JNK signaling pathway is required for stress-induced apoptosis. However, the molecular mechanism by which ceramide induces SAPK/JNK activation is unknown. Here we show that TAK1, a member of the mitogen-activated protein kinase kinase kinase family, is activated by treatment of cells with agents and stresses that induce an increase in ceramide. Ceramide itself stimulated the kinase activity of TAK1. Expression of a constitutively active form of TAK1 resulted in activation of SAPK/JNK and SEK1/MKK4, a direct activator of SAPK/JNK. Furthermore, expression of a kinase-negative form of TAK1 interfered with the activation of SAPK/JNK induced by ceramide. These results indicate that TAK1 may function as a mediator of ceramide signaling to SAPK/JNK activation.

A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.