Ras isoform abundance and signalling in human cancer cell lines.

The ubiquitously expressed major Ras isoforms: H-, K- and N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N- and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically K> or =N>>H. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

Ras proteins: paradigms for compartmentalised and isoform-specific signalling.

Ras GTPases mediate a wide variety of cellular processes by converting a multitude of extracellular stimuli into specific biological responses including proliferation, differentiation and survival. In mammalian cells, three ras genes encode four Ras isoforms (H-Ras, K-Ras4A, K-Ras4B and N-Ras) that are highly homologous but functionally distinct. Differences between the isoforms, including their post-translational modifications and intracellular sorting, mean that Ras has emerged as an important model system of compartmentalised signalling and membrane biology. Ras isoforms in different subcellular locations are proposed to recruit distinct upstream and downstream accessory proteins and activate multiple signalling pathways. Here, we summarise data relating to isoform-specific signalling, its role in disease and the mechanisms promoting compartmentalised signalling. Further understanding of this field will reveal the role of Ras signalling in development, cellular homeostasis and cancer and may suggest new therapeutic approaches.

Control of growth factor receptor dynamics by reversible ubiquitination.

Activated tyrosine kinase receptors acquire ubiquitin tags. Ubiquitination governs receptor down-regulation through interaction with components of the endosomal ESCRT (endosomal sorting complexes required for transport) machinery that shepherds receptors into luminal vesicles of multivesicular bodies en route to the lysosome. We have characterized two de-ubiquitinating enzymes that interact with components of this machinery. AMSH [associated molecule with the SH3 domain (Src homology 3 domain) of STAM (signal transducing adapter molecule)] shows specificity for Lys63- over Lys48-linked ubiquitin and may act to rescue receptors from taking the lysosomal pathway. In contrast, UBPY (ubiquitin-specific processing protease Y) does not discriminate between Lys48 and Lys63-linked chains and is required for lysosomal sorting.

Compartmentalization of Ras proteins.

The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effectors and exchange factors.

GTP-dependent segregation of H-ras from lipid rafts is required for biological activity.

Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins.

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4'-(maleimidomethyl)-cyclohexane-carboxamid o] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin-horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin-agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway.

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.

Localization of a class II phosphatidylinositol 3-kinase, PI3KC2alpha, to clathrin-coated vesicles.

We have analysed phosphatidylinositol 3-kinase activity associated with subcellular fractions prepared from rat brains. Phosphatidylinositol 3-kinase activity is not markedly enriched with synaptic vesicle purification; whilst the activity associated with the most pure fractions is inhibited at low concentrations of wortmannin (IC50 approximately 4-5 nM). In contrast, clathrin-coated vesicle (CCV) fractions showed increased enzyme activity compared to light membrane fractions from which they are purified. In addition to a wortmannin-sensitive activity, we also detected an activity that could only be inhibited at higher concentrations of wortmannin (IC50 approximately 400 nM), characteristic of certain class II enzymes (including phosphatidylinositol 3-kinase C2alpha) to be highly enriched in CCV fractions. Immunoblotting with an antibody raised against phosphatidylinositol 3-kinase C2alpha, confirmed that this enzyme is highly enriched in CCVs and displays an enrichment profile during the purification that mirrors enrichment of the low nanomolar wortmannin-insensitive activity. If the CCV purification protocol is adapted to favour nerve terminally derived vesicles, we find reduced levels of the C2alpha enzyme in the CCV fractions, suggesting that the enzyme may principally reside on vesicles associated with the cell body.

Glutamate uptake occurs at an early stage of synaptic vesicle recycling.

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.

Biological variability in a migrating isolate, the Tokelau Islands: Child growth in different environments.

The magnitude and rates of growth have been compared among two cross-sectional samples of Tokelau children on the basis of 26 anthropometric dimensions. One of the samples consisted of children living on the Tokelau Islands. The migrant sample consisted of children of Tokelau descent who were living in New Zealand. The comparison between samples suggest significant differences in the rate of linear growth at the younger ages. However, most differences were not significant among 17 year olds. The results support the hypothesis that even when the original environment is favorable, qualitative changes in the environment may affect the general pattern of growth. The effects of an accelerated growth pattern cannot be determined at this time.

The relationships between atopy, bronchial hyperresponsiveness, and a family history of asthma: a cross-sectional study of migrant Tokelauan children in New Zealand.

We have examined age-related changes in the association between nonspecific bronchial hyperresponsiveness (BHR) and atopy in 494 second-generation Polynesian migrant children, aged 5 to 15 years. BHR (provocative dose of methacholine, less than or equal to 7.8 mumol, causing a 20% fall in FEV1) was present in 125 children (25.3%). Atopy (skin wheal, greater than or equal to 4 mm diameter) was present in 157 children (32%). BHR associated with atopy demonstrated a constant age-related frequency in the 7- to 15-year-old children that was influenced by a family history (FH) of asthma (FH, 50%; no FH, 34%; p = 0.051). BHR not associated with atopy demonstrated a marked decreasing frequency with age from 25% in 5- to 7-year-old children to 3% in 13- to 15-year-old children and was uninfluenced by an FH of asthma. We conclude that the differences in the frequency of BHR with age, together with the genetic influence on BHR associated with atopy, compared with the findings in nonatopic children, indicate distinct heterogeneity in the pathogenesis of BHR in these Tokelauan children. These differences may be important for understanding the relationships between nonspecific BHR, atopy, and asthma in children.

Type 2 (non-insulin-dependent) diabetes mellitus, migration and westernisation: the Tokelau Island Migrant Study.

The migration of Tokelauans from a traditional atoll in the Pacific to urban New Zealand is associated with an increased prevalence and incidence of Type 2 (non-insulin-dependent) diabetes mellitus over the period 1968-1982. During the same period, a lesser but definite increase is seen among non-migrants in Tokelau. The age standardised prevalence rates rose from 7.5 and 11.7 to 10.8 and 19.9 per 100 respectively in the male and female migrants compared with an increase from 3.0 and 8.7 to 7.0 and 14.3 per 100 in the non-migrant males and females respectively. The incidence of diabetes is shown to be consistently higher in the migrants compared to the non-migrants giving relative risks of 1.5 in males and 1.9 in females. The factors most likely contributing to this difference, are changes to a higher calorie, high protein diet, higher alcohol consumption, a greater weight gain and altered levels of physical activity in the migrants. A number of populations in the Pacific have been shown to have a low rate of diabetes in their traditional setting, but may have a genetic predisposition for diabetes which responds to factors in the urban industrialised environment and life-style. The social and economic changes taking place in Tokelau are also clearly increasing the risk of diabetes. To reverse these trends and prevent the development of complications of Type 2 diabetes, it will be important to institute preventive programmes and to follow up the population in both environments for long-term outcomes, including mortality.

Blood pressure patterns and migration: a 14-year cohort study of adult Tokelauans.

The longitudinal relation between blood pressure changes and migration experience has been studied in a cohort of 654 adult Tokelauans through three survey periods between 1968 and 1982. Migration from a subsistence life-style on a Pacific atoll to an urbanized Western life-style in New Zealand is associated with increases in body mass in both men and women. Both the systolic and diastolic blood pressures of migrant men are significantly higher than would be expected in this cohort on the basis of age when compared with the nonmigrants. This is consistent with a rise around the time of migration to a level which is then maintained, with the diastolic pressures taking longer than the systolic pressures to respond to the migration stimuli. Most of this rise in blood pressure may be attributed to weight gain, but a significant part of the diastolic pressure excess remains unexplained. This pattern is not exhibited by the women, which may be a reflection of the sex roles in this Polynesian society. These findings indicate a need for new immigrants to be encouraged not to gain weight when confronted with new dietary choices.

Symptoms of asthma, methacholine airway responsiveness and atopy in migrant Tokelauan children.

503 migrant Tokelauan children between five and 15 years resident predominantly in Porirua and the Hutt Valley were surveyed as part of a study of asthma prevalence in a recently migrant population. The survey consisted of domiciliary interview with parents, physical examination, assessment of bronchial hyperresponsiveness and atopy, by allergen skin prick testing. Forty-three children (8%) had a history of wheezy breathing or asthma. 160 children (32%) had evidence of increased airway responsiveness defined as a PD20 (provocative dose of methacholine causing a less than or equal to 20% fall in FEV1, of less than or equal to 12.2 mumoL methacholine). Of the 43 children with a history of asthma, 40 (93%) had evidence of bronchial hyperresponsiveness, 36 (84%) were atopic and 35 (81%) had both bronchial hyperresponsiveness and atopy. Forty-five children (9%) were found to be wheezing on the day of examination only 16 of these had a history of wheezing. Twenty seven of the wheezing children demonstrated bronchial hyperresponsiveness and 22 of these were atopic. Of the 18 children wheezing but with no evidence of bronchial hyperresponsiveness only six were atopic. These contrasting findings suggest differences in the cause of symptoms among the children. Regional differences were observed for the prevalence of symptoms and signs of asthma, bronchial hyperresponsiveness and atopy. Hutt Valley Tokelauan children exhibited a higher prevalence than the Porirua children. Migrants to the Hutt Valley and Porirua are from different atolls, and these differences raise the possibility of a genetic influence on the development of asthma.

Migration and gout: the Tokelau Island migrant study.

The prevalence and 14 year incidence of clinical gout and its precursors were investigated in the Polynesian population of Tokelauans living in the Pacific basin, non-migrant Tokelauans living in their isolated atoll homeland being compared with migrant Tokelauans living in urban New Zealand. The age standardised prevalence of gout in Tokelauan men in New Zealand was higher than that in non-migrant Tokelauan men, being 21.0 and 19.5/1000 subjects at the beginning of the study and 51.0 and 14.6/1000 at the end of study, respectively. Migrant men in New Zealand aged under 55 had higher mean serum uric acid concentrations than non-migrant men of the same age. The prevalence of gout was low in women in both environments. The age standardised relative risk of developing gout between 1968 and 1982 was 9.0 times higher in the migrant men than in the non-migrant men. Age, serum uric acid concentration, serum cholesterol concentration, and self reported alcohol consumption at entry to the study were the best set of predictors of gout in men. Preventive strategies to change body mass, diet, and patterns of alcohol use need to be developed in this population.

24-h catecholamine excretion: relationships with age and weight.

24-hour urines were obtained from groups of volunteer subjects recruited from populations in the UK, USA, Nigeria and the South Pacific. Urines were collected during the course of normal daily activities. HPLC/electrochemical detection was used to measure concentrations of adrenaline and noradrenaline in each urine. Calculated 24-h output of catecholamines was tested for significant linear regression relationships with age, weight and 24-h urine volume in each population. Noradrenaline excretion tended to be positively related to age and to body wt. Adrenaline excretion tended to show little or no relationship with either age or body wt. Relationships based on catecholamine excretion/U creatinine were notably different to those based on absolute catecholamine excretion. Differences were observed in the nature and strength of the relationships in the different populations, indicating the likely importance of interactions with other factors.

Inter-population comparisons of catecholamine excretion.

Twenty-four hour urines were obtained from groups of male and female volunteers recruited from urban populations in developed and developing countries (UK, USA, Japan and Nigeria) an from agriculture-based Polynesian population (Tokelau Islands). The urines were collected on typical working days, with minimal disturbance to subjects' normal routines. High-performance liquid chromatography was used to determine catecholamine concentrations in each urine, providing a basis for calculation of total 24-hour excretion. Mean 24-hour excretion of noradrenaline and adrenaline was low in the Polynesians compared to the modern, urban groups, and low in women compared to men. Discussion of the results includes assessment of the possible influences of artefact, physical characteristics of the population samples, physical environment and social and psychological factors.

Rheumatic complaints in Tokelau. I. Migrants resident in New Zealand. The Tokelau Island migrant study.

The three Tokelau atolls are 8 degrees south of the equator. In 1966 the islands were involved in a severe hurricane which drew attention to overcrowding and led to resettlement of more than half the population in New Zealand. One thousand three hundred and eighty one migrants over 15 years old were examined in New Zealand in 1980 and 1981 for rheumatic complaints as part of a continuing assessment. Clinical criteria for osteoarthritis (COA), including crepitus in any joint and in the knee, showed an increase in prevalence with age and weight in both sexes. Partial correlation coefficient analysis showed an association of the number of affected joints or the severity of knee COA (COAK) with both age and weight. Stepwise regression showed that age was the best predictor of both COA and COAK scores. Weight had predictive value only for COAK and only in women. Using the tracking method, previous high and/or increasing weight was related to COAK observed at this assessment. Heberden nodes increased with age and were more prevalent in women but were not associated with weight. Low back, dorsal and neck pain showed no association with age or sex. Low back pain was associated with weight. Joint pain following injury occurred in 15.4% of men. Gout, more common in men, was the only frequent inflammatory arthritis found. Two definite cases of rheumatoid arthritis (RA) were identified and four had criteria 1 and 2 for the New York criteria.

Rheumatic complaints in Tokelau. II. A comparison of migrants in New Zealand and non-migrants. The Tokelau Island migrant study.

The migration of 1381 Tokelauans 15 years and over to New Zealand leaving 811 in Tokelau, provided a unique opportunity to test centuries of speculation on the impact of environment on rheumatic disease. There was no change in all rheumatic complaints. The migrant men had more gout, joint pain following injury, and neck pain. The migrant women had more dorsal back pain. There was no change in the prevalence of clinically defined osteoarthritis (COA) despite positive associations with weight and higher weights in the migrants. Rheumatoid arthritis was infrequent in both populations. Low back pain was common but was not more common in migrants, though compensation payments are readily available in New Zealand and are not in the Islands.