The B cell-specific major raft protein,Raftlin, is necessary for the integrity of lipid raft and BCR signal transduction

Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identification of proteins that co-purified with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.     

Lymphoid signal transduction mechanisms linked to cellular prion protein.

The normal cellular isoform of the prion protein (PrPC) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed widely, including in lymphoid cells. We compared lectin-induced mitogenesis and selected cell signaling pathways in splenocytes from wild-type BALB/c mice and Zrch Prnp0/0 (PrP0/0) mice bred on a BALB/c background for more than 10 generations. 3H-thymidine incorporation induced by concanavalin A (Con A) or phytohemagglutinin (PHA) was significantly reduced in PrP0/0 splenocytes, most prominently early in activation (24 and 48 h). Con A activation in PrP0/0 splenocytes was associated with differences in the phosphorylation (P) patterns of protein kinase C (PKC alpha/beta, but not delta) and the PKC downstream effectors p44/42MAPK (mitogen-activated protein kinase). P-PKC and P-MAPK profiles were similar in wild-type and PrP0/0 splenocytes following PMA treatment, indicating that the ability of these 2 enzymes to be phosphorylated is not impaired in the absence of PrPC. Con A-induced calcium fluxes, monitored by indo-1 fluorescence, were equivalent in PrP0/0 and PrP+/+ splenocytes, suggesting that calcium-dependent mechanisms are not directly implicated in the differential phosphorylation patterns or mitotic responses. Our data indicate that PrP0/0 splenocytes display defects in upstream or downstream mechanism(s) that modulate PKCalpha/beta phosphorylation, which in turn affects its capacity to regulate splenocyte mitosis, consistent with a role for PrPC in immune function.     

Cellular mechanisms of signal transduction for neurotrophins

The molecular cloning of new neuroactive growth factors and their receptors has greatly enhanced our understanding of important interactions among receptors and singnaling molecules. These studies have begun to illuminate some of the mechanisms that allow for specificity in neuronal signaling. Model cell systems, such as the PC-12 pheochromocytoma cell line, express receptors for these different neurotirophic factors, leading to comparisons of signaling pathways for these factors. Upon binding their ligands, these receptors undergo phosphorylation on tyrosine residues, which directs their interaction with signaling proteins containing src homology (SH2) domains, sequences that mediate associations with tyrosine-phosphorylated proteins. These SH2 proteins translate the tyrosine kinase activity of receptors into downstream events that result in the specific cellular response. Investigations such as these have revealed that molecular specificity in signaling pathways may arise from combinatorial diversity in interactions between receptors and key regulatory proteins.     

Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB

The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya. In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P(II) signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status. We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB-dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB. Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen-sufficient situations. The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status. The results also offer new insights into the evolution of P(II) proteins and a rationale for their trigonal symmetry.     

Approximations and their consequences for dynamic modelling of signal transduction pathways

Signal transduction is the process by which the cell converts one kind of signal or stimulus into another. This involves a sequence of biochemical reactions, carried out by proteins. The dynamic response of complex cell signalling networks can be modelled and simulated in the framework of chemical kinetics. The mathematical formulation of chemical kinetics results in a system of coupled differential equations. Simplifications can arise through assumptions and approximations. The paper provides a critical discussion of frequently employed approximations in dynamic modelling of signal transduction pathways. We discuss the requirements for conservation laws, steady state approximations, and the neglect of components. We show how these approximations simplify the mathematical treatment of biochemical networks but we also demonstrate differences between the complete system and its approximations with respect to the transient and steady state behavior.     

Receptor tyrosine kinase substrates: src homology domains and signal transduction.

Among the intracellular milieu of proteins are molecules with defined biochemical functions that serve as substrates for ligand-activated tyrosine kinase receptors. It seems likely that some of these substrate molecules are elements of a critical signaling pathway used by growth factors to control cell proliferation and subverted by oncogenes to deregulate this process. Although the process of cell growth and division is relatively slow compared with other hormonally regulated responses, homeostasis in a human being requires approximately 20 x 10(6) cell divisions per second for the renewal of various cell populations. This review summarizes the present understanding of tyrosine kinase substrates that seem likely to have key roles in the signal transduction pathway that regulates cell proliferation. This includes structural features of these molecules, the influence of tyrosine phosphorylation on their functions, the biological roles of these proteins, and the capacity of these substrates to associate with activated receptor tyrosine kinases.     

Perfringolysin O association with ordered lipid domains: implications for transmembrane protein raft affinity

Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) β-barrel. PFO is believed to interact with liquid ordered lipid domains (lipid rafts). Because the origin of TM protein affinity for rafts is poorly understood, we investigated PFO raft affinity in vesicles having coexisting ordered and disordered lipid domains. Fluorescence resonance energy transfer (FRET) from PFO Trp to domain-localized acceptors indicated that PFO generally has a raft affinity between that of LW peptide (low raft affinity) and cholera toxin B (high raft affinity) in vesicles containing ordered domains rich in brain sphingomyelin or distearoylphosphatidylcholine. FRET also showed that ceramide, which increases exposure of cholesterol to water and thus displaces it from rafts, does not displace PFO from ordered domains. This can be explained by shielding of PFO-bound cholesterol from water. Finally, FRET showed that PFO affinity for ordered domains was higher in its non-TM (prepore) form than in its TM form, demonstrating that the TM portion of PFO interacts unfavorably with rafts. Microscopy studies in giant unilamellar vesicles confirmed that PFO exhibits intermediate raft affinity, and showed that TM PFO (but not non-TM PFO) concentrated at the edges of liquid ordered domains. These studies suggest that a combination of binding to raft-associating molecules and having a rigid TM structure that is unable to pack well in a highly ordered lipid environment can control TM protein domain localization. To accommodate these constraints, raft-associated TM proteins in cells may tend to locate within liquid disordered shells encapsulated within ordered domains.     

缺氧诱导因子1与缺氧信号转导机制

缺氧诱导因子1(HIF-1)不仅作为维持氧自稳平衡的核心调控因子,调控一系列缺氧相关基因的表达,而且在感受缺氧,传递缺氧信号的过程中发挥重要作用。氧依赖的羟化酶的发现,证明胞内氧浓度直接调控HIF-1α亚基的表达,为揭示缺氧信号调控HIF-1表达的分子机制提供了有力的证据。     

BioNetGen: software for rule-based modeling of signal transduction based on the interactions of molecular domains

BioNetGen allows a user to create a computational model that characterizes the dynamics of a signal transduction system, and that accounts comprehensively and precisely for specified enzymatic activities, potential post-translational modifications and interactions of the domains of signaling molecules. The output defines and parameterizes the network of molecular species that can arise during signaling and provides functions that relate model variables to experimental readouts of interest. Models that can be generated are relevant for rational drug discovery, analysis of proteomic data and mechanistic studies of signal transduction.     

Signalling to translation: how signal transduction pathways control the protein synthetic machinery

Recent advances in our understanding of both the regulation of components of the translational machinery and the upstream signalling pathways that modulate them have provided important new insights into the mechanisms by which hormones, growth factors, nutrients and cellular energy status control protein synthesis in mammalian cells. The importance of proper control of mRNA translation is strikingly illustrated by the fact that defects in this process or its control are implicated in a number of disease states, such as cancer, tissue hypertrophy and neurodegeneration. Signalling pathways such as those involving mTOR (mammalian target of rapamycin) and mitogen-activated protein kinases modulate the phosphorylation of translation factors, the activities of the protein kinases that act upon them and the association of RNA-binding proteins with specific mRNAs. These effects contribute both to the overall control of protein synthesis (which is linked to cell growth) and to the modulation of the translation or stability of specific mRNAs. However, important questions remain about both the contributions of individual regulatory events to the control of general protein synthesis and the mechanisms by which the translation of specific mRNAs is controlled.     

Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction

Lateral mobility and spatial organization of proteins within the plasma membrane are likely to mediate the initial events coordinating T cell activation. Lipid rafts, distinct cholesterol/sphingolipid-rich membrane microdomains, provide a mechanism for this regulation by concentrating or excluding signaling proteins. We demonstrate in peripheral blood T cell lymphoblasts that immediate early phosphotyrosine signal transduction through the TCR complex is functionally dependent on a distinct population of lipid rafts. Specifically, cholesterol extraction destabilizes the membrane microdomains containing Lck, while the rafts containing the adapter protein linker for activation of T cells remain intact. Heterogeneity in the partitioning of these proteins in resting cells was confirmed by immunoelectron microscopy. After T cell activation, both Lck and the linker for activation of T cells colocalize to 50-100 nm microdomains in the plasma membrane, indicating that sequestration of these proteins into distinct lipid rafts may function to regulate the initiation of T cell signal transduction.