Nuclear lipid metabolism in NEST: Nuclear envelope signal transduction

Summary There is increasing evidence that nuclear lipid metabolism in NEST is an important new component in signal transducing networks and as a result, this metabolism is beginning to attract more attention. While agonistinduced nuclear lipid metabolism adds further complexity to the ever increasing array of signal transduction components, it also provides further avenues by which nuclear activities may be regulated. Identification of the coupling mechanisms, regulation, and physiological roles of nuclear lipid metabolism represents a new and exciting area of research which will have a broad impact in our understanding of signal transduction pathways.     

Phosphotyrosine-binding domains in signal transduction

Protein phosphorylation provides molecular control of complex physiological events within cells. In many cases, phosphorylation on specific amino acids directly controls the assembly of multi-protein complexes by recruiting phospho-specific binding modules. Here, the function, structure, and cell biology of phosphotyrosine-binding domains is discussed.     

Genes involved in cytokinin signal transduction

Although cytokinin plays a central role in plant development, our knowledge about the signal transduction pathway initiated by this plant hormone is fragmentary. By randomly introducing enhancer elements into theArabidopsis genome throughAgrobacterium-mediated transformation, 5 cytokinin independent mutant calli (cki1-1, −2, −3, −4 andcki2) were obtained. These mutants exhibit typical cytokinin responses, including rapid proliferation, chloroplast differentiation, shoot induction and inhibition of root formation, in the absence of cytokinin. TheCKl1 gene encodes a product similar to the sensor histidine kinases of two-component systems, and its overexpression in plants induces typical cytokinin responses (Kakimoto 1996). Here I report that overexpression of this gene did not alter the auxin reqirement ofArabidopsis. Another mutant,many shoots, which was also identified on the same screening, produced many adventitious shoots on cotyledons, petioles and true leaves. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.     

Nuclear phospholipase C: involvement in signal transduction

During the past years, several independent laboratories have highlighted the presence of nuclear signaling pathways based on lipid hydrolysis, which are not a mere duplication of those occurring at the plasma membrane. Among the enzymes of the cycle, nuclear phosphoinositide-specific phospholipase C (PI-PLC) has been analyzed quite extensively. In this context, PI-PLCbeta1 appears to play a key role as a check point in the G1 phase of the cell cycle. It has also been shown that its activation and/or up-regulation is upon the control of type 1 insulin-like growth factor receptor (IGF-R) in both mouse fibroblast and myoblasts, suggesting that its signaling activity is essential for the normal behavior of the cell, at least in culture. The recent discovery of a possible involvement of the deletion of PI-PLCbeta1 gene in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) in humans strengthens the contention that nuclear PI-PLC signaling is essential for physiological processes such as cell growth and differentiation. Even though PI-PLCbeta1 is present and does not translocate to eukaryotic nuclei, this organelle, even though only in some conditions contains also PI-PLCgamma1 which acts not only as a PI-PLC but also as guanine nucleotide exchange factor (GEF) for PI 3-kinase enhancer (PIKE) and is somehow linked to PI 3-kinase (PI3K) activity. Also members of PI-PLCdelta family are shuttling from the nucleus to the cytoplasm and return and are possibly involved in the control of cell growth. We must also take into account the presence in the nucleus of other phospholipases such as phospholipase A2 (PLA2) and phospholipase D (PLD), which also exert a signaling activity upon external stimuli. On the whole this review highlights the latest development in the PI-PLC cycle in the nucleus, which in terms of activation, regulation and down-stream targets differs substantially from that located at the plasma membrane.     

Modelling protein functional domains in signal transduction using Maude

Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.     

脯氨酸参与植物开花信号转导途径

作为一种渗透调节物质,人们对脯氨酸已经有了很多的研究,然而脯氨酸在生命体生长发育中的作用还知之甚少。为了研究脯氨酸在生长发育中的作用,筛选得到了拟南芥的脯氨酸抗性突变体kao2。突变体在不同浓度脯氨酸培养基上均表现出抗性,但是突变体中脯氨酸的吸收没有显著变化,降解关键基因表达也没有增多。Tail-PCR进行突变基因克隆的结果表明,T-DNA插入位点位于AtKAO2(At2g32440)的第一个外显子,且插入导致AtKAO2基因不再表达。拟南芥突变体库中订购的kao2-2同样表现出脯氨酸抗性表型。此外,kao2表现出胁迫敏感表型。kao2表现出明显的晚花表型,并且赤霉素的添加能够部分挽救晚花表型。而kao2突变体中开花抑制因子FLC表达升高,而开花促进基因FT的表达降低。研究在脯氨酸与开花信号转导途径之间建立了联系,为脯氨酸对开花途径的信号作用提供了新的证据,为以后的研究提供了新的材料。     

Novel domains of the prokaryotic two-component signal transduction systems

The archetypal two-component signal transduction systems include a sensor histidine kinase and a response regulator, which consists of a receiver CheY-like domain and a DNA-binding domain. Sequence analysis of the sensor kinases and response regulators encoded in complete bacterial and archaeal genomes revealed complex domain architectures for many of them and allowed the identification of several novel conserved domains, such as PAS, GAF, HAMP, GGDEF, EAL, and HD-GYP. All of these domains are widely represented in bacteria, including 19 copies of the GGDEF domain and 17 copies of the EAL domain encoded in the Escherichia coli genome. In contrast, these novel signaling domains are much less abundant in bacterial parasites and in archaea, with none at all found in some archaeal species. This skewed phyletic distribution suggests that the newly discovered complexity of signal transduction systems emerged early in the evolution of bacteria, with subsequent massive loss in parasites and some horizontal dissemination among archaea. Only a few proteins containing these domains have been studied experimentally, and their exact biochemical functions remain obscure; they may include transformations of novel signal molecules, such as the recently identified cyclic diguanylate. Recent experimental data provide the first direct evidence of the participation of these domains in signal transduction pathways, including regulation of virulence genes and extracellular enzyme production in the human pathogens Bordetella pertussis and Borrelia burgdorferi and the plant pathogen Xanthomonas campestris. Gene-neighborhood analysis of these new domains suggests their participation in a variety of processes, from mercury and phage resistance to maintenance of virulence plasmids. It appears that the real picture of the complexity of phosphorelay signal transduction in prokaryotes is only beginning to unfold.     

Calmodulin is involved in heat shock signal transduction in wheat

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.     

Nuclear inositol lipid signaling and its potential involvement in malignant transformation

Growth, differentiation, and apoptosis of eukaryotic cells are mediated by extremely complex signaling pathways and a high degree of coordination is required for regulating cell proliferation.In multicellular organisms homeostasis is achieved through signal transduction events. If these homeostatic mechanisms are interrupted, a disease, such as cancer, may ensue. Lipid second messengers, particularly those derived from polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signaling molecules are important components of signaling pathways operating within the nucleus. Recent findings are starting to elucidate how the nuclear phosphoinositide cycle is regulated and what down-stream molecules are targeted through this cycle. In this review, we shall summarize the most updated data about inositol lipid-dependent nuclear signaling pathways that might have a relevance for the development of cancer. In the near future, this knowledge might also prove to have relevance for the diagnosis and treatment of the neoplastic disease.     

Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction

We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BCtheta. The BCtheta-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (approximately 1.0) than the BCtheta-unbound population in raft fractions (approximately 0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling cascade, are also contained in the BCtheta-bound membranes. On the other hand, CD3epsilon, CD3zeta and Zap70 are localized in the BCtheta-unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3epsilon, CD3zeta and Zap70 are recruited to the BCtheta-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCtheta-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells, which could be a core domain controlling the TCR signalling cascade.     

Investigation of signal transduction pathways involved in melanoma cell spreading

Integrins are a major family of heterodimeric adhesion receptors that are responsible for anchoring cells to extracellular matrix and they also can initiate intracellular signal pathways. Here parental and alpha 4-expressing human malignant melanoma cell lines were used to study the effect of protein kinase C (PKC), protein tyrosine kinases (PTKs) and intracellular Ca2+ on alpha 4 beta 1-mediated cell spreading on VCAM-1. Incubation of melanoma cells with PKC inhibitor inhibited alpha 4 beta 1-mediated melanoma cell spreading completely. Effect of intracellular Ca2+ on melanoma cell spreading was also investigated by non-phorbol ester tumor promotor, thapsigargin, which blocks the ability of the endoplasmic reticulum to replenish stocks of calcium which naturally leak out into the cytosol leading to a transient increase in concentration of intracellular calcium. The results showed that alpha 4 beta 1-mediated spreading was also required intracellular calcium involvement. However, in the presence of PTKs inhibitor melanoma cells showed long, thin dendiritic projections compared to control cells. Previously, data was obtained from immunofluorescense experiments showed that after genistein treatment, alpha 4-expressing cells exhibited considerable amounts of alpha 4 integrin and PTKs in both the focal contact points as well as over the whole cell. PTKs inhibitor did not have any effect on alpha 4-expressing cells spreading. This could be related to the amount of the PTKs present in these cells.     

受体内化和核转位参与的细胞信息传递

近年来,对在物质信息的细胞内传递过程中进一步的认识,发现这些物质除了可经第二信使系统引起生物效应外,尚可通过激动剂介导的受体内化和核转位反应直接引起配体特异性的核基因表达改变。本文对受体内化和核转位过程及其影响因素作一概述。     

Functional roles of glycosphingolipids in signal transduction via lipid rafts

The formation of glycosphingolipid (GSL)-cholesterol microdomains in cell membranes has been proposed to function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are postulated to be involved in GPI-anchored protein signaling via src-family kinase. Here, the functional roles of GSLs in signal transduction mediated by the microdomains are discussed. Antibodies against GSLs co-precipitate GPI-anchored proteins, src-family kinases and several components of the microdomains. Antibody-mediated crosslinking of GSLs, as well as that of GPI-anchored proteins, induces a rapid activation of src-family kinases and a transient increase in the tyrosine phosphorylation of several substrates. Enzymatic degradation of GSLs reduces the activation of src-family kinase and tyrosine phosphorylation by antibody-mediated crosslinking of GPI-anchored protein. Furthermore, GSLs can also modulate signal transduction of immunoreceptors and growth factor receptors in the microdomains. Thus, GSLs have important roles in signal transduction mediated by the microdomains.     

The membrane and lipids as integral participants in signal transduction: lipid signal transduction for the non-lipid biochemist

Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction. However, lipids serve a variety of roles in signal transduction. They act as ligands that activate signal transduction pathways as well as mediators of signaling pathways, and lipids are the substrates of lipid kinases and lipid phosphatases. Cell membranes are the source of the lipids involved in signal transduction, but membranes also constitute lipid barriers that must be traversed by signal transduction pathways. The purpose of this review is to explore the magnitude and diversity of the roles of the cell membrane and lipids in signal transduction and to highlight the interrelatedness of families of lipid mediators in signal transduction.