Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product

THE superfamily of low molecular mass GTP-binding proteins, for which the ras proteins are prototypes, has been implicated in the regulation of diverse biological activities including protein trafficking, secretion, and cell growth and differentiation. One member of this family, CDC42Hs (originally referred to as Gp or G25K), seems to be the human homologue of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. A second S. cerevisiae protein, CDC24, which is known from complementation studies to act with CDC42Sc to regulate the development of normal cell shape and the selection of nonrandom budding sites in yeast, contains a region with sequence similarity to the dbl oncogene product. Here we show that dbl specifically catalyses the dissociation of GDP from CDC42Hs and thereby qualifies as a highly selective guanine nucleotide exchange factor for the GTP-binding protein. Although guanine nucleotide exchange activities have been previously described for other members of the Ras-related GTP-binding protein family, this is the first demonstration, to our knowledge, of the involvement of a human oncogenic protein in catalysing exchange activity.     

Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation.

Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.     

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.     

Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors

Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases-the master regulators of the actin cytoskeleton. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology.     

Phospholipase C gamma 1 is a physiological guanine nucleotide exchange factor for the nuclear GTPase PIKE

Phospholipase C gamma 1 (PLC-gamma 1) hydrolyses phosphatidylinositol-4,5-bisphosphate to the second messengers inositol-1,4,5-trisphosphate and diacylglycerol. PLC-gamma 1 also has mitogenic activity upon growth-factor-dependent tyrosine phosphorylation; however, this activity is not dependent on the phospholipase activity of PLC-gamma 1, but requires an SH3 domain. Here, we demonstrate that PLC-gamma 1 acts as a guanine nucleotide exchange factor (GEF) for PIKE (phosphatidylinositol-3-OH kinase (PI(3)K) enhancer). PIKE is a nuclear GTPase that activates nuclear PI(3)K activity, and mediates the physiological activation by nerve growth factor (NGF) of nuclear PI(3)K activity. This enzymatic activity accounts for the mitogenic properties of PLC-gamma 1.     

Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein-GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf.GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.     

Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.

The fungal metabolite brefeldin A is a powerful tool for investigating membrane traffic in eukaryotic cells. The effects of brefeldin A on traffic are partly explained by its ability to prevent binding of cytosolic coat proteins onto membranes. The non-clathrin coatomer complex binds reversibly to Golgi membranes in a GTP-controlled cycle. The low-molecular-mass GTP-binding protein ADP-ribosylation factor (ARF), which also associates reversibly with Golgi membranes, is required for coatomer binding and probably accounts for the control by guanine nucleotide of the coatomer-membrane interaction. Brefeldin A prevents the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane, but the nature of this interaction has not been established. Here we demonstrate that Golgi membranes can specifically catalyse the exchange of GTP onto ARF and that brefeldin A prevents this function.     

Reaction dynamics between the photonuclease triplet-excited vitamin-K3 and guanine nucleotide

Nanosecond laser pulse photolysis was used to investigate the excited-state dynamics of the photonuclease vitamin-K3 (VK3). A spectral band peaked at 285 nm was newly observed and attributed to the triplet-excited state of VK3. In studying the photochemistry between the photonuclease VK3 and guanine nucleotide (dGMP), we obtained spectroscopic evidence for the photo-initiated formation of both VK3 radical anion and dGMP radical cation, and thus proved the charge transfer reaction between the triplet-excited VK3 and the ground-state dGMP.     

Uncoupling of cardiac muscarinic and beta-adrenergic receptors from ion channels by a guanine nucleotide analogue

Guanine nucleotide binding proteins, interchangeably called N or G proteins, seem to be the primary signal-transducing components of various agonist-induced cell membrane functions. In the heart, G proteins have been implicated in beta-adrenergic modulation of the slow inward Ca2+ current. We have investigated the role of G proteins in muscarinic activation of an inwardly rectifying, acetylcholine (ACh)-induced K+ current (IACh), and beta-adrenergic activation of an (isoprenaline)-induced Ca2+ current (Isi). Here we report that intracellular application of the non-hydrolysable GTP analogue 5'-guanylylimidodiphosphate (GppNHp) brought about an agonist-induced, antagonist-resistant, persistent activation of IACh and Isi. This functional uncoupling of channel from receptor suggests that the muscarinic receptor and the IACh channel are separate molecular structures. Membrane conductance responses to sequential activation of muscarinic and beta-adrenergic receptors demonstrate that in contrast to the muscarinic inhibition of Isi, muscarinic stimulation of IACh is mediated by a G protein via a pathway that does not involve adenylate cyclase. Taken together, the results support the notion that agonist is required to induce GppNHp binding and/or activation of the G proteins. Once triggered by agonist, the control system remains maximally activated, thereby transforming the cell so that it no longer responds to subsequent homologous receptor-mediated signals.     

Ca2+ release from endoplasmic reticulum is mediated by a guanine nucleotide regulatory mechanism

Ca2+ accumulation and release from intracellular organelles is important for Ca2+-signalling events within cells. In a variety of cell types, the active Ca2+-pumping properties of endoplasmic reticulum (ER) have been directly studied using chemically permeabilized cells. The same preparations have been extensively used to study Ca2+ release from ER, in particular, release mediated by the intracellular messenger inositol 1,4,5-trisphosphate (InsP3). So far, these studies and others using microsomal membrane fractions have revealed few mechanistic details of Ca2+ release from ER, although a recent report indicated that InsP3-mediated Ca2+ release from liver microsomes may be dependent on GTP. In contrast to the latter report, we describe here the direct activation of a specific and sensitive guanine nucleotide regulatory mechanism mediating a substantial release of Ca2+ from the ER of cells of the neuronal cell line N1E-115. These data indicate the operation of a major new Ca2+ gating mechanism in ER which is specifically activated by GTP, deactivated by GDP, and which appears to involve a GTP hydrolytic cycle.     

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity and appear to interact with G-proteins, perhaps by causing phosphorylation of them. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predisposition to infection. We show here that experimentally-induced type I diabetes leads to the loss of expression of Gi in rat liver. As it has been suggested that Gi may couple receptors to K+-channels as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.     

Functional reconstitution of purified muscarinic receptors and inhibitory guanine nucleotide regulatory protein

Muscarinic receptors trigger several different responses including an increase in concentration of cyclic GMP, a decrease in cyclic AMP concentration, breakdown of polyphosphoinositides and changes in ion permeability. It is not yet clear whether these reactions occur sequentially or independently and which directly coupled to the muscarinic receptor. Several lines of evidence indicate that muscarinic receptors in many, if not all, cell types are coupled to the inhibitory guanine nucleotide regulatory protein (Ni or Gi) of adenylate cyclase. To provide direct evidence for this coupling, we have reconstituted muscarinic receptors purified from porcine brain with Ni purified from rat brain in a phospholipid vesicle. Here, we report that the GTPase activity of Ni is stimulated by carbachol. This action is blocked by the simultaneous addition of atropine and is not observed when the Ni protein is ADP-ribosylated. We conclude that one function of the muscarinic receptor is the activation of Ni.     

孤芳自赏的美丽与哀愁——陈染《私人生活》中的自恋情结

20世纪90年代女性小说创作热潮中,陈染以其独特的思维方式和生命体验坚持个人话语的言说,坚持个性化写作,塑造了一批反传统伦理常规的女性形象。其长篇小说《私人生活》讲述了主人公倪拗拗的身心成长历程,通过对主人公孤独自闭、同性恋倾向、异性恋中的主体意识等方面的分析,可以见出小说文本中的自恋情结。     

Control of phyllotaxy by the cytokinin-inducible response regulator homologue ABPHYL1

Phyllotaxy describes the geometric pattern of leaves and flowers, and has intrigued botanists and mathematicians for centuries. How these patterns are initiated is poorly understood, and this is partly due to the paucity of mutants. Signalling by the plant hormone auxin appears to determine the site of leaf initiation; however, this observation does not explain how distinct patterns of phyllotaxy are initiated. abphyl1 (abph1) mutants of maize initiate leaves in a decussate pattern (that is, paired at 180 degrees), in contrast to the alternating or distichous phyllotaxy observed in wild-type maize and other grasses. Here we show that ABPH1 is homologous to two-component response regulators and is induced by the plant hormone cytokinin. ABPH1 is expressed in the embryonic shoot apical meristem, and its spatial expression pattern changes rapidly with cytokinin treatment. We propose that ABPH1 controls phyllotactic patterning by negatively regulating the cytokinin-induced expansion of the shoot meristem, thereby limiting the space available for primordium initiation at the apex.     

Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins

Several human tumour cell lines contain genes that can transform NIH 3T3 cells into malignant cells. Certain genes have been classified as members of the ras oncogene family, namely, Ha-ras, Ki-ras or N-ras. The proteins encoded by the ras family are generally small (Ha-ras, for example, encodes a protein of molecular weight 21,000 named p21), and are associated with the inner surface of the plasma membrane. The only known biochemical property common to all forms of the ras proteins is the ability to bind guanine nucleotides, a property which may be closely related to the transforming ability of ras proteins. A GTP-dependent, apparent autophosphorylation (on threonine 59) activity has been identified only in the case of the v-Ha-ras protein. Although the role of these biochemical activities in the transformation process remains unclear, we have initiated studies to determine the possible biochemical interactions of ras proteins with other membrane components. We report here the evidence that epidermal growth factor enhances the guanine nucleotide binding activity of activated c-Ha-ras or v-Ha-ras p21, and phosphorylation of v-Ha-ras p21, suggesting that some mitogenic growth factors may regulate those activities.     

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.     

微波催化合成环己基三氯硅烷的研究

以环己烯和三氯氢硅为原料,氯铂酸为催化剂,经微波催化硅氢加成反应合成了环己基三氯硅烷.研究了催化剂用量、微波诱导和微波加热对反应的影响,确立了最佳的合成条件:用二甲苯作溶剂,催化剂用量取初始反应体系中浓度120 mg.L-1,微波加热功率为100 W,加热时间为1 h,三氯氢硅的转化率为68%,选择性为98.9%.     

头孢三嗪聚乙二醇催化合成研究

研究了聚乙二醇催化合成头孢三嗪的工艺,探讨了聚乙二醇分子量,用量,反应温度及反应时间等对缩合产物收率的影响。当用聚乙二醇－８００作为相转移催化剂,在二氯甲烷－水溶剂中于０℃反应４￣６ｈ收率９４％。     

表面结构对催化剂选择性的影响

本文以萘氧化制苯酐催化剂为例,研究确定了催化剂选择性与表面孔结构的关系,並获得了适宜孔结构的最佳工艺条件。