Search

Systemic inflammation arising from the organismal distribution of pathogen-associated molecular patterns is a major cause of clinical morbidity and mortality. Herein we report a critical and previously unrecognized in vivo role for germinal center kinase (GCK, genome nomenclature: map4k2), a mammalian Sterile 20 (STE20) orthologue, in PAMP signaling, and systemic inflammation. We find that disruption of gck in mice strongly impairs PAMP-stimulated macrophage cytokine and chemokine release and renders mice resistant to endotoxin-mediated lethality. Bone marrow transplantation studies show that hematopoietic cell GCK signaling is essential to systemic inflammation. Disruption of gck substantially reduces PAMP activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) via reduced activation of the MAPK-kinase-kinases (MAP3Ks) mixed lineage kinases (MLKs)-2 and -3. Extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB) activation are largely unaffected. Thus, GCK is an essential PAMP effector coupling JNK and p38, but not ERK or NF-kappaB to systemic inflammation.

Cell matrix adhesion is required for cell proliferation and survival. Here we report that mutation by gene targeting of the cytoplasmic tail of [[beta].sub.1] integrin leads to defective proliferation and survival both in vivo and in vitro. Primary murine embryonic fibroblasts (MEFs) derived from mutant homozygotes display defective cell cycle coupled to impaired activation of the FAK-PI3K-Akt and Rac-JNK signaling pathways. Expression in homozygous MEFs of a constitutively active form of Rac is able to rescue proliferation, survival, and JNK activation. Moreover, although showing normal Erk phosphorylation, mutant cells fail to display Erk nuclear translocation upon fibronectin adhesion. However, expression of the constitutively activated form of Rac restores Erk nuclear localization, suggesting that adhesion-dependent Rac activation is necessary to integrate signals directed to promote MAPK activity. Altogether, our data provide the evidence for an epistatic interaction between the [[beta].sub.1] integrin cytoplasmic domain and Rac, and indicate that this anchorage-dependent signaling pathway is crucial for cell growth control.

Stress effects in U937 human monoblastic leukemia and bovine aortic endothelial cells were studied to determine the effect of relating the sphingomyelin and SAPK/JNK signalling systems in the induction of cell death or apoptosis. C2-ceramide was found to effect apoptosis as measured by DNA-specific fluorochrome bisbenzimide and confirmed by TUNEL assay. SAPK activation by C2-ceramide and sphingomyelinase was forty times than the control. Stress-induced apoptosis was reported to be initiated by ceramide but with an operational SAPK/JNK cascade as a requirement.

The germinal centre kinase (GCK), a human STE20 homologue, has the ability to activate the mammalian stress-activated protein kinase (SAPK) pathway. SAPKs, protein kinases of the ERK type, are activated in situ by inflammatory stimuli and they in turn are probably responsible for stimulating the transactivation function of c-Jun(super 5-7). The activation of SAPK pathway by GCK demonstrates the conservation mechanism of the eukaryotic signalling process and also reveals the physiological function of a mammalian Ste20.

A newly identified protein kinase, termed stress-activated protein-kinase (SAPK)/ERK kinase-1 (SEK1), mediates the in vitro and in vivo activation of the SAPKs, which are c-Jun amino-terminal protein kinases activated by cellular stresses. A structural relation exists between SEK1 and the MAP kinase kinases. SEK1 forms a signalling cascade that correlates cellular stress agonists and the c-Jun transcription factor. SAPK activation is inhibited by an inactive SEK1 mutant via extracellular stimuli and this action does not affect the MAPK pathway.

Protein kinase p54 is isolated from cycloheximide-treated rats and belongs to a family of extracellularly regulated kinases. Proline-directed p54 is more active in the phosphorylation of the c-Jun transactivating domain. P54, c-Jun N-terminal kinases, activated by heat shock and tumor necrosis factor (TNF)-alpha, are called stress-activated protein kinases (SAPK). TNF-alpha activation of SAPKs occurs through the signalling pathway involving spingomyelin-hydrolysis.

Ras regulation of Raf kinase activity depends on the c-Raf CR 1 domain. Raf-1 and Ras can bind directly in vitro and in situ. Test results imply that the active Ras polypeptide targets the c-Raf-1 kinase. The Ras protein appears to regulate the activation state of a set of cytoplasmic protein Ser/Thr kinases. The c-Raf cysteine finger's role suggests that homologous domains in other proteins may enable those proteins to interact directly with Ras.

The mitogen-activated protein (MAP) kinase can be catalyzed into a related form, MAP kinase-kinase (MAPK-K), which takes part in developing v-raf-transformed cells, raf being a transforming oncogene. MAPK-K can be rendered inactive by phosphatase 2A; c-Raf-1, a protooncogene product, can restore MAPK-K to the active state. These findings show that c-Raf-1 is an immediate upstream activator of MAPK-K and that MAPK-K is the first physiological substrate of a protooncogene product to be discovered.

Analysis of NIH3T3 cell sublines expressing MEK kinase-1 (MEKK) shows that MEKK induction activates the stress-activated protein kinases (SAPKs). A new signalling cascade is regulated by MEKK upon phosphorylation of the SAPK activator, SEK1, which phosphorylates and stimulates SAPK. MEKK is similar to kinases mediating yeast mating signal cascades and is involved in vitro MEK activation.

I. INTRODUCTION Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. All eukaryotic cells possess multiple MAPK pathways, each of which is [...]

Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element-binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3β (GSK3β) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K+ (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K+ channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3β inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3β mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3β inhibitors Li+ and/or CHIR-99021, Li+ increased IKACh, and Li+ and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3β activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3β as a new therapeutic target for diabetic autonomic neuropathy. [ABSTRACT FROM AUTHOR]