Phosphorylation-induced Conformational Changes in Rap1b: ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP*

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser¹⁷⁹ at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser¹⁷⁹ phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.Rap1b, a member of the Ras superfamily of small G proteins, is a GTPase that acts as a molecular on/off switch for the transduction of several external stimuli by alternating from an inactive GDP-bound to an active GTP-bound state (1, 2). Rap1 activation is mediated by several second messengers, growth factors, cytokines, and cell adhesion molecules. The steady-state level of Rap1-GTP is tightly regulated by a family of guanine nucleotide exchange factors that catalyze the otherwise slow dissociation of GDP (i.e. activation) and GTPase-activating proteins, which stimulate the rather slow intrinsic GTPase catalytic activity (i.e. inactivation) (3). GTP binding is coupled to conformational changes in two well defined regions, the switch I (residues 30–40) and switch II (residues 60–76) domains, responsible for high affinity interaction with effector molecules (4, 5) and thus downstream signal transduction.cAMP is one among several pathways leading to Rap1 activation (6). cAMP exerts both mitogenic and anti-mitogenic responses in different cell types, and Rap1 activation is required downstream of cAMP in both scenarios (7, 8). Elevation of intracellular cAMP levels activates cAMP-dependent protein kinase (PKA)⁴ and Epac (exchange protein activated by cAMP), a Rap guanine nucleotide exchange factor (9). Expression of Rap1b in cells where cAMP is mitogenic is associated with an increase in cAMP-mediated G₁/S phase entry (7, 10), and both biochemical events, Rap activation and phosphorylation at Ser¹⁷⁹, are synergistically required for this action (11).PKA substrates able to modulate Rap1 activity (i.e. Src/C3G recruitment and GTPase-activating protein) were recently reported (12, 13). However, the role of PKA-dependent Rap1 phosphorylation at Ser¹⁷⁹ is still unknown. Rap1 phosphorylation does not affect its overall intracellular localization, its basal GTP/GDP exchange reaction, its intrinsic rate of GTP hydrolysis, or its ability to be stimulated by a cytosolic Rap GTPase-activating protein (10); however, several reports suggest that Rap1 phosphorylation is able to modulate its association with some binding partners, namely cytochrome b₅₅₈ (14) and Raf1 (15). The mechanism by which a modification of Ser¹⁷⁹ at the C-terminal end of the molecule affects the regions involved with effector interaction at its N terminus is for the moment unclear.In this study, we report a global assessment of the effects of Ser¹⁷⁹ phosphorylation on conformational change/mobility analyzed by hydrogen/deuterium exchange mass spectrometry (DXMS). The results are consistent with an allosteric effect of the C terminus (containing Ser¹⁷⁹) to the switch loops/effector domain.     

Children with Moderate Acute Malnutrition with No Access to Supplementary Feeding Programmes Experience High Rates of Deterioration and No Improvement: Results from a Prospective Cohort Study in Rural Ethiopia

BackgroundChildren with moderate acute malnutrition (MAM) have an increased risk of mortality, infections and impaired physical and cognitive development compared to well-nourished children. In parts of Ethiopia not considered chronically food insecure there are no supplementary feeding programmes (SFPs) for treating MAM. The short-term outcomes of children who have MAM in such areas are not currently described, and there remains an urgent need for evidence-based policy recommendations.MethodsWe defined MAM as mid-upper arm circumference (MUAC) of ≥11.0cm and <12.5cm with no bilateral pitting oedema to include Ethiopian government and World Health Organisation cut-offs. We prospectively surveyed 884 children aged 6–59 months living with MAM in a rural area of Ethiopia not eligible for a supplementary feeding programme. Weekly home visits were made for seven months (28 weeks), covering the end of peak malnutrition through to the post-harvest period (the most food secure window), collecting anthropometric, socio-demographic and food security data.ResultsBy the end of the study follow up, 32.5% (287/884) remained with MAM, 9.3% (82/884) experienced at least one episode of SAM (MUAC <11cm and/or bilateral pitting oedema), and 0.9% (8/884) died. Only 54.2% of the children recovered with no episode of SAM by the end of the study. Of those who developed SAM half still had MAM at the end of the follow up period. The median (interquartile range) time to recovery was 9 (4–15) weeks. Children with the lowest MUAC at enrolment had a significantly higher risk of remaining with MAM and a lower chance of recovering.ConclusionsChildren with MAM during the post-harvest season in an area not eligible for SFP experience an extremely high incidence of SAM and a low recovery rate. Not having a targeted nutrition-specific intervention to address MAM in this context places children with MAM at excessive risk of adverse outcomes. Further preventive and curative approaches should urgently be considered.     

Anemia among Primary School Children in Eastern Ethiopia

Background

Anemia during childhood impairs physical growth, cognitive development and school performance. Identifying the causes of anemia in specific contexts can help efforts to prevent negative consequences of anemia among children. The objective of this study was to assess prevalence and identify correlates of anemia among school children in Eastern Ethiopia.

Methods

A cross sectional study was conducted from January 2012 to February 2012 in Kersa, Eastern Ethiopia. The study included randomly selected primary school students. Hemoglobin concentration was measured using a Hemocue haemoglobinometer. A child was identified as anemic if the hemoglobin concentration was <11.5 g/dl for children (5–11 yrs) and < 12 g/dl for child older than 12 years age. Poisson regression model with robust variance was used to calculate prevalence ratios.

Result

The overall prevalence of anemia was 27.1% (95% CI: 24.98, 29.14): 13.8% had mild, 10.8% moderate, and 2.3% severe anemia. Children with in the age group of 5-9 years (APR, 1.083; 95% CI, 1.044- 1.124) were at higher risk for anemia. Paternal education (Illiterate, 1.109; 1.044 - 1.178) was positively associated with anemia. Children who had irregular legume consumption (APR, 1.069; 95% CI, 1.022 -1.118) were at higher risk for anemia.

Conclusion

About a quarter of school children suffer from anemia and their educational potential is likely to be affected especially for those with moderate and severe anemia. Child age, irregular legume consumption, and low paternal schooling were associated with anemia. Intervention programmes aimed to reduce anemia among school children are crucial to ensure proper growth and development of children.     

Mapping the nucleotide and isoform-dependent structural and dynamical features of Ras proteins

Ras GTPases are conformational switches controlling cell proliferation, differentiation, and development. Despite their prominent role in many forms of cancer, the mechanism of conformational transition between inactive GDP-bound and active GTP-bound states remains unclear. Here we describe a detailed analysis of available experimental structures and molecular dynamics simulations to quantitatively assess the structural and dynamical features of active and inactive states and their interconversion. We demonstrate that GTP-bound and nucleotide-free G12V H-ras sample a wide region of conformational space, and show that the inactive-to-active transition is a multiphase process defined by the relative rearrangement of the two switches and the orientation of Tyr32. We also modeled and simulated N- and K-ras proteins and found that K-ras is more flexible than N- and H-ras. We identified a number of isoform-specific, long-range side chain interactions that define unique pathways of communication between the nucleotide binding site and the C terminus.