Role of protein kinase Cdelta in UV-B-induced apoptosis of macrophages in vitro

We have previously reported that murine peritoneal macrophages exposed to ultraviolet B (UV-B; 100 mJ/cm2) undergo apoptosis, as indicated by alterations in cell morphology, caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, DNA fragmentation, sustained activation of p38/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) and inactivation of p42/44 MAPKs. It is now reported that macrophages undergoing UV-B-induced apoptosis show enhanced expression of protein kinase Cdelta (PKCdelta) in a time-dependent manner. Pretreatment of macrophages with PKCdelta-specific inhibitor rottlerin prior to the UV-B irradiation inhibits activation of caspase-3, PARP cleavage, DNA fragmentation and release of intracellular Ca2+. Inhibition of PKCdelta also blocks the sustained activation of p38 and JNK MAPKs as well as inactivation of p42/44 MAPKs. PKCalpha and PKCbeta1 expression also increases during UV-B-induced apoptosis in macrophages. Inhibition of these two isoforms with Go6976 slightly suppresses caspase-3 activation, PARP cleavage, DNA fragmentation and release of intracellular Ca2+, but has no effect on the sustained activation of p38/JNK MAPKs or inactivation of p42/44 MAPKs. It is, therefore, suggested that activation of PKCdelta might play an important role in the UV-B-induced apoptosis and that specific activated isoforms of PKC may have distinct functions in cell death.     

Formyl peptide receptor like 1 differentially requires mitogen-activated protein kinases for the induction of glial fibrillary acidic protein and interleukin-1alpha in human U87 astrocytoma cells

Mitogen-activated protein kinases (MAPKs) are not only pivotal mediators of signal transduction but they also regulate diverse biological processes ranging from survival, proliferation and differentiation to apoptosis. By using human U87 astrocytoma and transfected FPRL1/CHO cells, we have demonstrated that activation of FPRL1 with WKYMVM effectively phosphorylated JNK and ERK. Interestingly, p38 MAPK activation was only seen with FPRL1/CHO cells. The MAPK phosphorylations in response to WKYMVM were blocked by WRW(4) (a selective FPRL1 antagonist), but not cyclosporine H (a well-known FPR antagonist). The key signaling intermediates in the MAPK pathways were also delineated. G(i)/G(o) proteins, Src family tyrosine kinases, but not phosphatidylinositol-3 kinase, protein kinase C and calmodulin-dependent kinase II, were required to transmit signals from FPRL1 toward JNK, ERK and p38 MAPK. Furthermore, phospholipase Cbeta was distinctively involved in the regulation of JNK but not the other MAPKs. Importantly, WKYMVM-stimulated U87 cells triggered noticeable increases in glial fibrillary acidic protein (GFAP) and interleukin-1alpha (IL-1alpha), which are correlated with reactive astrocytosis. In contrast, GFAP expression was not altered following stimulation with N-formyl-methionyl-leucyl-phenylalanine. Moreover, inhibitions of G(i)/G(o) proteins and JNK completely abolished both GFAP and IL-1alpha upregulations by FPRL1, while blockade of the MEK/ERK cascade exclusively suppressed the GFAP production. Consistently, overexpression of MEK1 and constitutively active JNKK in U87 cells led to ERK and JNK activation, respectively, which was accompanied with markedly increased GFAP production. We have thus identified a possible linkage among FPRL1, MAPKs, astrocytic activation and the inflammatory response.     

NKIATRE is a novel conserved cdc2-related kinase

The mitogen-activated protein kinases (MAPKs) and the cyclin-dependent kinases (CDKs) are key mediators of cell proliferation in response to extracellular signals. Recent additions to each of these families and the identification of kinases with structural features of both have provided insights into fundamental processes, such as cell division and differentiation. To identify novel serine kinases with features of MAPKs or CDKs, a degenerate PCR-based amplification approach was undertaken. The 57- and 52-kDa isoforms of a novel protein kinase, termed NKIATRE, were molecularly cloned from rat brain and jejunum cDNA libraries. Like the MAPKs, NKIATRE has a Thr-Xaa-Tyr motif in kinase subdomain VIII. NKIATRE also shows close homology to the cyclin-dependent kinase class of protein kinases and the cdc2-related kinases NKIAMRE, KKIALRE, and KKIAMRE, containing both conserved inhibitory phosphorylation sites and a putative cyclin-binding domain. Two isoforms of NKIATRE that differ in their carboxy-terminal ends have been identified. A functional nuclear localization signal is specific to the longer 57-kDa alpha isoform. Sequence similarity to the putative human tumor suppressor gene NKIAMRE, which is lost in leukemic patients with chromosome 5q deletions, suggests that NKIATRE may have a role in restricting cell growth or maintaining differentiation.     

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.     

Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3

The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.     

c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-beta (SAPKbeta) binds and phosphorylates the neuronal microtubule regulator SCG10.

The neuronal growth-associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule-destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine-threonine-directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen-activated protein (MAP) kinases, c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKbeta. JNK3/SAPKbeta phosphorylation occurs at Ser-62 and Ser-73, residues that result in reduced microtubule-destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKbeta activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.     

丝裂原活化蛋白激酶(MAPK)生物学功能的结构基础

丝裂原活化蛋白激酶 (MAPK)是生物体内重要的信号转导系统之一 ,能对广泛的细胞外刺激发生反应 .蛋白激酶的空间构象是其功能的重要决定因素 .对MAPK蛋白结构的研究表明 ,MAPK的结构与功能之间具有密切的关系 .尽管MAPK各亚族的结构非常相似 ,但也存在着一些差异 ,这些差异是不同亚族对不同的细胞外刺激产生特异性反应的结构基础 .某些关键性结构 ,例如Loop12 ,在MAPK对上游激酶的作用、下游底物的选择以及亚细胞定位中都具有重要作用 .进一步深入研究MAPK的空间结构 ,探讨MAPK的生物学功能与其空间构象之间的关系 ,对于开发新的MAPK通路抑制剂用于治疗某些严重疾病有着重要的临床意义     

Mitogen-activated protein kinase signaling in plants under abiotic stress

Mitogen-activated protein kinase cascade is evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. This cascade consists essentially of three components, a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK connected to each other by the event of phosphorylation. These kinases play various roles in intra- and extra-cellular signaling in plants by transferring the information from sensors to responses. Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as responses to various stresses. MAPK signaling has also been associated with hormonal responses. In plants, MAP kinases are represented by multigene families and are involved in efficient transmission of specific stimuli and also involved in the regulation of the antioxidant defense system in response to stress signaling. In the current review we summarize and investigate the participation of MAPKs as possible mediators of various abiotic stresses in plants.Key words: abiotic stress, cross talk, mitogen-activated protein kinases, heat map, MAPK signaling, signal transduction, stress signaling     

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.     

Mitogen-activated protein kinases and their role in regulation of cellular processes

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes connecting cell-surface receptors to critical regulatory targets within cells. The three major MAPK cascades are known, the extracellular signal-regulated protein kinase (ERK) cascade, c-Jun amino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade and p38-MAPK cascade. This paper is focused on characterization of these MAPK cascades in terms of their distribution and biological role in some pathological processes (apoptosis, hypertrophy) with a special orientation on the role of MAPKs in cardiovascular system during ischemia/reperfusion.     

c-Jun N-terminal kinase (JNK) signaling: Recent advances and challenges

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.     

Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress

Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra-cellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.     

Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.

Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.     

The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.     

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, Sin1alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin1 polyclonal antibody showed that full-length Sin1 and several smaller isoforms are widely expressed. Sin1 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 Sin1alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1alpha 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.     

Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration

We have observed that the vasoactive peptide endothelin-1 is a potent inducer of migration of primary human brain-derived microvascular endothelial cells. By blocking signal transduction pathways with specific inhibitors, and using dominant negative mutant infections, we have demonstrated that multiple pathways are involved in endothelin-1-induced migration. Absolutely required for migration are protein tyrosine kinase Src, Ras, protein kinase C (PKC), phosphatidylinositol 3-kinase, ERK, and JNK; partial requirements were exhibited by cAMP-activated protein kinase and p38 kinase. Partial elucidation of the signal transduction sequences showed that the MAPKs ERK, JNK, and p38 are positioned downstream of both PKC and cAMP-activated protein kinase in the signal transduction scheme. The results show that human brain endothelial cell migration has distinct characteristics, different from cells derived from other vascular beds, or from other species, often used as model systems. Furthermore, the results indicate that endothelin-1, secreted by many tumors, is an important contributor to tumor-produced proangiogenic microenvironment. This growth factor has been associated with increased microvessel density in tumors and is responsible for endothelial cell proliferation, migration, invasion, and tubule formation. Because many signal transduction pathways investigated in this study are potential or current targets for anti-angiogenesis therapy, these results are of critical importance for designing physiological antiangiogenic protocols. signal transduction; angiogenesis; microvessels; vasoactive peptides