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.     

Bcr encodes a GTPase-activating protein for p21rac

More than thirty small guanine nucleotide-binding proteins related to the ras-encoded oncoprotein, termed Ras or p21ras, are known. They regulate many fundamental processes in all eukaryotic cells, such as growth, vesicle traffic and cytoskeletal organization. GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins, leading to down-regulation of the active GTP-bound form. For p21ras, two GAP proteins are known, rasGAP and the neurofibromatosis (NF1) gene product. There is evidence that rasGAP may also be a target protein for regulation by Ras and be involved in downstream signalling. We have purified a GAP protein for p21rho, which is involved in the regulation of the actin cytoskeleton. Partial sequencing of rhoGAP reveals significant homology with the product of the bcr (breakpoint cluster region) gene, the translocation breakpoint in Philadelphia chromosome-positive chronic myeloid leukaemias. We show here that the carboxy-terminal domains of the bcr-encoded protein (Bcr) and of a Bcr-related protein, n-chimaerin, are both GAP proteins for the Ras-related GTP-binding protein, p21rac. This result suggest that Bcr could be a target for regulation by Rac and has important new implications for the role of bcr translocations in leukaemia.     

PDGF induction of tyrosine phosphorylation of GTPase activating protein

The cascade of biochemical events triggered by growth factors and their receptors is central to understanding normal cell-growth regulation and its subversion in cancer. Ras proteins (p21ras) have been implicated in signal transduction pathways used by several growth factors, including platelet-derived growth factor (PDGF). These guanine nucleotide-binding Ras proteins specifically interact with a cellular GTPase-activating protein (GAP). Here we report that in intact quiescent fibroblasts, both AA and BB homodimers of PDGF rapidly induce tyrosine phosphorylation of GAP under conditions in which insulin and basic fibroblast growth factor (bFGF) are ineffective. Although GAP is located predominantly in the cytosol, most tyrosine-phosphorylated GAP is associated with the cell membrane, the site of p21ras biological activity. These results provide a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signalling system.     

Involvement of p21ras in activation of extracellular signal-regulated kinase 2.

Many growth factors upon stimulation of their receptors induce the activity of extracellular signal-regulated kinases, ERKs, also known as MAP kinases. Several of these growth factors also activate the ras proto-oncogene product, p21ras (Ras), by stimulating the conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. We have shown that direct introduction of p21ras oncoprotein into cells in the absence of growth factors activates ERKs within five minutes, which indicates that normal p21ras may be involved in the activation of ERKs by growth factors. Here we use a recombinant vaccinia virus expressing an interfering mutant of p21ras, RasAsn17, to investigate this question. In NIH3T3 cells that overexpress the insulin receptor, this recombinant virus inhibits insulin-induced activation of ERK2 completely, but there is no inhibition of insulin-induced activation of phosphatidylinositol-3-kinase. In rat-1 cells the recombinant virus inhibited ERK2 activity induced by platelet-derived growth factor (PDGF) but not by phorbol ester. We conclude that p21ras mediates insulin- and PDGF-induced activation of ERK2.     

Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i)

Many receptors for neuropeptides and hormones are coupled with the heterotrimeric G(i) protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPK) cascade through both the alpha- and betagamma-subunits of G(i). The betagamma-subunit activates the ERK/MAPK cascade through tyrosine kinase. Constitutively active G(alpha)i2 (gip2) isolated from adrenal and ovarian tumours transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism. The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes Ras function. Here we show that a novel isoform of Rapl GTPase-activating protein, called rap1GAPII, binds specifically to the alpha-subunits of the G(i) family of heterotrimeric G-proteins. Stimulation of the G(i)-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the alpha-subunit of G(i) activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.     

The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product

About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins and control cell proliferation by regulating signal transduction pathways at the plasma membrane. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site, strongly implicating GAP as the biological target for regulation by p21.     

Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.     

A new family of RhoGEFs activates the Rop molecular switch in plants

In plants, the small GTP-binding proteins called Rops work as signalling switches that control growth, development and plant responses to various environmental stimuli. Rop proteins (Rho of plants, Rac-like and AtRac in Arabidopsis thaliana) belong to the Rho family of Ras-related GTP-binding proteins that turn on signalling pathways by switching from a GDP-bound inactive to a GTP-bound active conformation. Activation depends on guanine nucleotide exchange factors (GEFs) that catalyse the otherwise slow GDP dissociation for subsequent GTP binding. Although numerous RhoGEFs exist in animals and yeasts, no Rop-specific GEFs have yet been identified in plants and so Rop activation has remained elusive. Here we describe a new family of RhoGEF proteins that are exclusive to plants. We define a unique domain within these RopGEFs, termed PRONE (plant-specific Rop nucleotide exchanger), which is exclusively active towards members of the Rop subfamily. It increases nucleotide dissociation from Rop more than a thousand-fold and forms a tight complex with nucleotide-free Rop. RopGEFs may represent the missing link in signal transduction from receptor kinases to Rops and their identification has implications for the evolution of the Rho molecular switch.     

Bioorganic synthesis of lipid-modified proteins for the study of signal transduction

Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.     

Association between GTPase activators for Rho and Ras families.

The ras-related low-molecular-mass GTPases participate in signal transduction involving a variety of cellular functions, including cell-cycle progression, cellular differentiation, cytoskeletal organization, protein transport and secretion. The cycling of these proteins between GTP-bound and GDP-bound states is partially controlled by GTPase activating proteins (GAPs) which stimulate the intrinsic GTP-hydrolysing activity of specific GTPases. The ras GTPase-activating protein (Ras-GAP) forms a complex with a second protein, p190 (M(r) 190,000), in growth-factor stimulated and tyrosine-kinase transformed cells. At its carboxy-terminal end, p190 contains a region that is conserved in the breakpoint cluster region, n-chimaerin, and Rho-GAP. Each of these three proteins exhibits GAP activity for at least one member of the rho family of small GTPases. We have tested recombinant p190 protein for GAP activity on GTPases of the ras, rho and rab families, and show here that p190 can function as a GAP specifically for members of the rho family. Consequently, the formation of a complex between Ras-GAP and p190 in growth-factor stimulated cells may allow the coupling of signalling pathways that involve ras and rho GTPases.     

Co-capping of ras proteins with surface immunoglobulins in B lymphocytes

Cellular ras genes encode a family of membrane-associated proteins (p21ras) that bind guanine nucleotide and possess a low intrinsic GTPase activity. The p21ras proteins are ubiquitously expressed in mammalian cells and are thought to be involved in a growth-promoting signal transduction pathway; their mode of action, however, remains unknown. The ligand-induced movement of cell-surface receptors seems to be a primary event in the transduction of several extracellular signals that control cell growth and differentiation. In B lymphocytes, surface immunoglobulin receptors crosslinked by antibody or other multivalent ligands form aggregates called patches, which then collect into a single assembly, a cap, at one pole of the cell. This process constitutes the initial signal for the activation of a B cell. Here we show by immunofluorescence microscopy that p21ras co-caps with surface immunoglobulin molecules in mouse splenic B lymphocytes. In contrast, no apparent change in the distribution of p21ras occurs during the capping of concanavalin A receptors. The redistribution of p21ras is apparent at the early stages (patching) of immunoglobulin capping and is inhibited by metabolic inhibitors and the cytoskeleton-disrupting agents colchicine and cytochalasin D. The distribution of another membrane-associated guanine nucleotide-binding regulatory protein, the Gi alpha subunit, is not affected by surface immunoglobulin capping. These findings demonstrate that p21ras can migrate in a directed manner along the plasma membrane and suggest that p21ras may be a component of the signalling pathway initiated by the capping of surface immunoglobulin in B lymphocytes.     

Involvement of p21ras in Xenopus mesoderm induction.

During early vertebrate embryogenesis, mesoderm is specified by a signal emanating from prospective endoderm. This signal can respecify Xenopus prospective ectoderm as mesoderm, and can be mimicked by members of the fibroblast growth factor and transforming growth factor-beta families. In other systems, the p21c-ras proto-oncogene product has been implicated in signal transduction for various polypeptide growth factors. We report here that a dominant inhibitory ras mutant blocks the mesoderm-inducing activity of fibroblast growth factor and activin, as well as the endogenous inducing activity of prospective endoderm. A constitutively active ras mutant partially mimics these activities. These results indicate that p21ras may have a central role in the transduction of the mesoderm inductive signal. Basic fibroblast growth factor and activin have emerged as candidates for endogenous mesoderm-inducing molecules. The character of the mesoderm induced by these two factors is overlapping but distinct when assessed both by histological and molecular criteria. The signal transduction pathways used during induction by these factors are unknown. We used messenger RNA microinjection of Xenopus eggs to express a dominant inhibitory mutant ras, p21(Asn 17)Ha-ras, in cells competent to respond to inducing factors to examine the role of p21ras in this response. This mutant, which has a reduced affinity for GTP relative to GDP, blocks a variety of mitogenic signals in 3T3 fibroblasts as well as the differentiation of pheochromocytoma cells in response to nerve growth factor.     

Tumour maintenance is mediated by eNOS

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.     

Structural determinants for GoLoco-induced inhibition of nucleotide release by Galpha subunits

Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Galpha subunits and maintain G-protein subunit dissociation in the absence of Galpha activation. Here we describe the structural determinants of GoLoco activity as revealed by the crystal structure of Galpha(i1) GDP bound to the GoLoco region of the 'regulator of G-protein signalling' protein RGS14. Key contacts are described between the GoLoco motif and Galpha protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Galpha to make direct contact with the GDP alpha- and beta-phosphates. The structural organization of the GoLoco Galpha(i1) complex, when combined with supporting data from domain-swapping experiments, suggests that the Galpha all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco Galpha interactions.     

细胞周期末期促进复合物亚基CDC16Hs与DNA双链断端修复蛋白Ku80相互作用

CDC16Hs是细胞周期末期促进复合物(APC)的亚基．利用基于LexA的酵母双杂交系统,把它作为诱饵蛋白筛选人胎脑文库,发现它与DNA双链断端修复蛋白Ku80的羧基端相互作用．CDC16Hs和全长Ku80的结合通过pull down实验在体外得到验证．     

Spatio-temporal images of growth-factor-induced activation of Ras and Rap1.

G proteins of the Ras family function as molecular switches in many signalling cascades; however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses and how the signals for differentiation and survival are distinguished by neuronal cells.     

Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK.

The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation. They do so by binding to downstream effector proteins. Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs). The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP. This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins. Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK. Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex. The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding.     

The p66shc adaptor protein controls oxidative stress response and life span in mammals

Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.     

Reaction path of protein farnesyltransferase at atomic resolution

Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. FTase inhibitors are currently in clinical trials for the treatment of cancer. Here we present a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. A structural model for the transition state consistent with previous mechanistic studies was also constructed. The processive nature of the reaction suggests the structural basis for the successive addition of two prenyl groups to Rab proteins by the homologous enzyme geranylgeranyltransferase type-II. Finally, known FTase inhibitors seem to differ in their mechanism of inhibiting the enzyme.     

The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways

Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.