PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits ...of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.
The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass ...of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.
Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of ...the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in
S. pombe
1, 2 and a PI-4-kinase in
D. melanogaster
3) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.
Understanding the magnitude of and uncertainty around soil carbon flux (SCF) is important in light of California’s efforts to increase SCF (from the atmosphere to soils) for climate change ...mitigation. SCF depends, to a great extent, on how soils are managed. Here, we summarize the results of an elicitation of soil science and carbon cycle experts aiming to characterize understanding of current SCF in California’s cropland and rangeland, and how it may respond to alternative management practices over time. We considered four cropland management practices—biochar, compost, cover crops, and no-till—and two rangeland management practices, compost and high-impact grazing. Results across all management practices reveal underlying uncertainties as well as very modest opportunities for soil carbon management to contribute meaningfully to California’s climate mitigation. Under median scenarios, experts expect all the surveyed management practices to reverse SCF from negative to positive, with direct carbon additions via biochar and compost offering the best potential for boosting the soil carbon pool.
Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola ...CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC
of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely ...unknown. Here we show that messenger RNA (mRNA) expression of
and
, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for
, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the
promoter region abrogates E2F1-mediated induction of a
luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the
promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of
with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.
Most research on cultural stressors and alcohol has focused on intercultural stressors. Continuing to exclude intracultural stressors (e.g., intragroup marginalization) from alcohol research will ...yield a biased understanding of the experiences of Hispanics living in a bicultural society. As we amass more studies on intracultural stressors, research will be needed to identify mutable sociocultural factors that may mitigate the association between intracultural stressors and alcohol. To address these limitations, we examined the association between intragroup marginalization and alcohol use severity and the extent to which gender and bicultural self-efficacy may moderate this association. A convenience sample of 200 Hispanic emerging adults ages 18-25 (men = 101, women = 99) from Arizona (n = 99) and Florida (n = 101) completed a cross-sectional survey. Data were analyzed using hierarchical multiple regression and moderation analyses. Higher intragroup marginalization was associated with higher alcohol use severity. Gender functioned as a moderator whereby intragroup marginalization was associated with higher alcohol use severity among men, but not women. Also, higher social groundedness functioned as a moderator that weakened the association between intragroup marginalization and alcohol use severity. Role repertoire did not function as a moderator. Our findings are significant because they enhance the reliability of the association between intragroup marginalization and alcohol use severity, and the moderating effect of gender in this respective association. This emerging line of research suggests that alcohol interventions targeting Hispanics may have a significant limitation by not accounting for intracultural stressors.
E2F transcription factors control diverse biological processes through regulation of target gene expression. However, the mechanism by which this regulation is established, and the relative ...contribution of each E2F member are still poorly defined. We have investigated the role of E2F2 in regulating cellular proliferation. We show that E2F2 is required for the normal G0/G1 phase because targeted disruption of the E2F2 gene causes T cells to enter S phase early and to undergo accelerated cell division. A large set of E2F target genes involved in DNA replication and cell cycle progression (such as Mcm's, cyclins and Cdc2a) that are silent in G0 and typically transcribed late in G1 phase are already actively expressed in quiescent T cells and MEFs lacking E2F2. The classic E2F activators, E2F1 and E2F3, are largely dispensable for this process because compound loss of E2F1-/- and E2F2-/- produces a comparably shortened G0/G1 phase, with early S phase entry. Likewise, shRNA knockdown of E2F3 does not alter significantly the E2F2-/- phenotype. Chromatin immunoprecipitation analysis indicates that in wild-type cells the promoters of the aberrantly early-transcribed genes are occupied by E2F2 in G0, suggesting a direct role for E2F2 in transcriptional repression. We conclude that E2F2 functions to transcriptionally repress cell cycle genes to establish the G0 state.
Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the ...insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.
The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants we identified the ...phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, we examined PtdInsP-binding of Pbl and the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pblΔPH mutants. Taken together, our results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration.
► The RhoGEF Pebble (Pbl) is required for cytokinesis and mesoderm migration. ► pi5K59B was identified in a screen for regulators of migration in pbl mutants. ► PtdIns(4,5)P2s are enriched at the front of mesodermal cells, and enhance migration. ► The PH domain of Pbl binds PtdIns(4,5)P2 and is required for cortical localisation. ► The PH domain is essential for mesoderm migration.
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