c-Cbl is not required for ERK1/2-dependent degradation of BimEL

Bim_EL the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim_EL. It has been proposed that the RING finger protein Cbl binds to Bim_EL and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim_EL is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim_EL turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim_EL is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim_EL does not involve its tyrosine phosphorylation; indeed, Bim_EL is not a tyrosine phosphoprotein. Furthermore, Bim_EL fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim_EL turnover proceeds normally in Cbl-containing or Cbl−/− fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim_EL turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.     

Range size, dispersal and niche breadth in the herbaceous flora of central England

1 It has been suggested that rare species differ from related common species in a variety of traits, including (among others) niche breadth and dispersal ability. We set out to test these ideas in the flora of central England.
2 We used two measures of range: number of hectads in Britain (national range) and number of 1 km² in a 3000-km² area of central England (local range). In each case we regressed phylogenetically independent contrasts in geographical range against contrasts in range of germination temperature (one aspect of fundamental niche breadth), terminal velocity of dispersule (a measure of wind dispersal capacity), seed weight and specialism index (a measure of the diversity of habitats exploited in central England). Seed weights and germination temperature were known for 263 species, specialism index for 261 and terminal velocity for 178.
3 Specialism index explained by far the largest part of the variance in local and national range. Seed weight, terminal velocity and germination temperature each explained only 2–4% of the variation in local range, and none of the variation in national range.
4 Most previous attempts to relate niche breadth and dispersal ability to range have concerned animals. There is little convincing published evidence for plants, and in the British herbaceous seed plants studied here the best predictor of range was diversity of habitats exploited. This pattern was independent of phylogeny.     

Cbl-mediated negative regulation of platelet-derived growth factor receptor-dependent cell proliferation. A critical role for Cbl tyrosine kinase-binding domain.

The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-alpha (PDGFRalpha) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRalpha signaling machinery strongly suggested that Cbl negatively regulates PDGFRalpha signaling. Here, we show that, similar to PDGFRalpha, selective stimulation of PDGFRbeta induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFRbeta, as observed with PDGFRalpha. We show that Cbl-dependent negative regulation of PDGFRalpha and beta results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.     

Systems approaches reveal that ABCB and PIN proteins mediate co-dependent auxin efflux

Members of the B family of membrane-bound ATP-binding cassette (ABC) transporters represent key components of the auxin efflux machinery in plants. Over the last two decades, experimental studies have shown that modifying ATP-binding cassette sub-family B (ABCB) expression affects auxin distribution and plant phenotypes. However, precisely how ABCB proteins transport auxin in conjunction with the more widely studied family of PIN-formed (PIN) auxin efflux transporters is unclear, and studies using heterologous systems have produced conflicting results. Here, we integrate ABCB localization data into a multicellular model of auxin transport in the Arabidopsis thaliana root tip to predict how ABCB-mediated auxin transport impacts organ-scale auxin distribution. We use our model to test five potential ABCB–PIN regulatory interactions, simulating the auxin dynamics for each interaction and quantitatively comparing the predictions with experimental images of the DII-VENUS auxin reporter in wild-type and abcb single and double loss-of-function mutants. Only specific ABCB–PIN regulatory interactions result in predictions that recreate the experimentally observed DII-VENUS distributions and long-distance auxin transport. Our results suggest that ABCBs enable auxin efflux independently of PINs; however, PIN-mediated auxin efflux is predominantly through a co-dependent efflux where co-localized with ABCBs.

Predicting the experimentally observed root-tip auxin distribution requires ABCBs to efflux auxin independently, whereas PINs predominantly mediate auxin efflux where co-localized with ABCBs.     

Prostacyclin and steroidogenesis in goat ovari cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 μg/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P₄) and estradiol-17β (E₂) productin by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10⁻⁶−10⁻³M) and indomethacin (100 ng−1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 μ/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 μg− 1 mg/ml) and dipyridamole (10⁻⁶−10⁻³M), when added alone or along with AA, did not effect steroid production. Up to 100 μg/ml of U-51605 (9,11-azoprosta-5, 13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGl₂) and its stable analog 6βPGl₁(0.01–10μg/ml) produced a dose-dependent increase in P₄ and E₂ production in all three cell types. Increase at 1 and 10μg/ml was significant in all cases. 6-keto-PGE₁ (an active metabolite of PGl₂ in certain systems) produced an increase in steroid production which was significant in theca at 1μg/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGf₁ alpha (stable metabolite of PGl₂) was without effect inthe present system. The lack of effect of PGl₂ at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolene. The present results indicate that AA- stimualted steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGl₂ though PGl₂ itself can positively modulate the steroid production.     

Characterization of paternity relationships in the mole rat <Emphasis Type="Italic">Spalax ehrenbergi</Emphasis> by microsatellite genotyping

Spalax ehrenbergi is a species complex of blind subterranean rodents distributed along the east Mediterranean region. We studied genetic relationships within and between S. ehrenbergi sib families using microsatellite genotyping. The upper-bound level of multiple-paternity rate in litters was estimated using a simulation model of breeding process. Our results show that the upper bound of multiple paternity in the studied population of S. ehrenbergi is <30% (P value 2.9%), with no detected cases of multiple paternity. Our analytical model was specifically designed for a situation in which the sibling identities are known but genetic data about their parents is unavailable. The genetic similarities between groups of individuals were also tested, and it was found that the distance between breeding nests is a factor influencing the genetic similarity between litters found in the nests.     

Heterozygote deficiency caused by a null allele at the bovine ARO23 microsatellite

Abstract

Non‐Mendelian inheritance and heterozygote deficiency were observed within the International Bovine Reference Panel (IBRP) when genotyped for AGC trinucleotide microsatellite ARO23. Chi square analysis showed a significant difference between the observed and predicted heterozygosity among 37 unrelated individuals. PCR reactions using an alternative primer designed to avoid a putative mismatch resulted in the appearance of an additional allele with a frequency of 0.30 and the restoration of Mendelian inheritance. Sequence analysis of this allele showed a cytosine insertion 2 bp from the 3’ end of the original priming site causing the failure of allele amplification. The presence of a segregating null allele may be suspected when heterozygote deficiency is observed.     

Novel Oncogenic Mutations of CBL in Human Acute Myeloid Leukemia That Activate Growth and Survival Pathways Depend on Increased Metabolism

Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs.     

The human kallikrein 10 promoter contains a functional retinoid response element

Human kallikrein 10 (hK10) protein is expressed in normal breast but is significantly downregulated in a majority of invasive breast cancers. Thus, understanding how hK10 expression is regulated is of substantial significance. In this study, we analyzed the promoter region of hK10 using a website software (TRANSFAC 3.0), which predicted three possible retinoic acid response elements (RAREs), RARE1 at -1041 (TGACCTCGTGATCC), RARE2 at -859 (TGACCTCCTATGA) and RARE3 at -765 (TGACCTCCTGTGA), each with a half-site of a canonical sequence (TGACCT; reverse complement AGGTCA). Using electrophoretic mobility shift assays and nucleotide competition analysis, as well as chromatin immunoprecipitation of the native hK10 promoter, we demonstrated specific binding of RXR only to RARE1. The functional importance of RARE in the hK10 promoter was demonstrated by retinoid induction of hk10 promoter-reporters; furthermore, mutation of RARE1 but not of RARE2 or RARE3 abolished the induction of the reporter. Finally, we demonstrated the induction of hK10 mRNA and protein expression upon retinoid treatment of cells. In view of the correlation of the downregulation of hK10 mRNA and protein with breast cancer progression, these findings suggest a potential approach to restore hK10 expression in cancer patients.