Jürgen Scheele

Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes.

E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.

Rap1A-Deficient T and B Cells Show Impaired Integrin-Mediated Cell Adhesion

ABSTRACT Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1−/− cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.

Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis

Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.

Kinetics of CO Ligation with Nitric-oxide Synthase by Flash Photolysis and Stopped-flow Spectrophotometry

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce ⋅NO and citrulline from l-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2–3 × 106 m −1 s−1) and another slow (2–4 × 104 m −1s−1). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, ∼50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s−1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.

TIME‐RESOLVED FLUORESCENCE STUDIES OF SPINACH CHLOROPLASTS–EVIDENCE FOR THE HETEROGENEOUS BIPARTITE MODEL

Abstract— Fluorescence lifetimes of spinach chloroplasts were measured with a modelocked dye laser and time‐correlated single photon counting. Information about energy transport and functional organization of the chloroplasts is revealed by such time‐resolved fluorescence studies. Quenching experiments using treatment with UV light or the chemical agent dibromothymoquinone are consistent with the notion that there is heterogeneity associated with PS II units and that such heterogeneity is reflected over the entire time range of fluorescence decay, not just in a single component. Phosphorylation experiments were also carried out which permit us to relate these kinetic studies to previous steady state observations.

KINETIC MODELING OF TIME‐RESOLVED FLUORESCENCE IN SPINACH CHLOROPLASTS

Abstract— Time‐resolved fluorescence decay profiles following picosecond excitation of plant chloro‐plasts or algae are now being measured with high precision and reproducibility. A number of suggestions have been put forward to explain the complex decay kinetics which are observed. In this paper three models are defined explicitly, solved rigorously, and compared in detail with experimental data. The complete description of energy transport in chloroplasts is no doubt very complicated, and only such quantitative comparisons will make it possible to decide which models are consistent with experiments. Of the three models examined, only the heterogeneous bipartite model has solutions which agree with commonly agreed upon features of the data. It offers, therefore, one starting point for more elaborate models and a guide to the design of further experiments.

Transcriptional profiling during the early differentiation of granulocyte and monocyte progenitors controlled by conditional versions of the E2a-Pbx1 oncoprotein.

The E2a–Pbxl oncoprotein of human pre-B cell leukemia prevents differentiation and maintains continued cell division in cultured myeloid progenitors. Previously, estrogen-dependent forms of E2a–Pbxl were generated that immortalized neutrophil (ECoM-G cells) or monocyte (ECoM-M cells) progenitors and that permitted their terminal differentiation upon estrogen withdrawal. Here, representational difference analysis (RDA) and Affymetrix array analysis are used to identify changes in gene expression that accompany the early differentiation of these cells. The promoters of these genes, whose expression changes upon E2a–Pbxl inactivation, integrate the biochemical mechanism through which E2a–Pbx1 arrests differentiation and maintains cell division. Inactivation of E2a–Pbxl caused the 10- to 80-fold up regulation of a small subset of myeloid differentiation genes (MRP8, Cnlp, NB1, Bactenecin, YM1, Stefin 1, Lipocortin, Lactoferrin, gp91 phox and Ly6-G) and a 10-fold down regulation of the TLE1 corepressor gene, as well as of a group of genes expressed in dividing cells (c-Myc, Nucleophosmin, Spermidine synthase, NOP56, Hnrpa1). Transcription of 97% of cellular genes, including 300 other transcription factor genes (21 Hox genes) and other myeloid genes, varied less than 3-fold, with most varying less than 50%. Therefore, E2a–Pbxl prevents transcription and maintains the cell cycle by a specific rather than a global transcriptional mechanism. Monocyte progenitors were distinguished by persistent expression of IRF8 and of a category of other genes characterized as “interferon-stimulated” (ISG15, ISG20, Ifit1, Ifi202a, Ifi203, Ifi204, Ifi204-related, IRF7 and Ly6-E.1), as well as by the upregulation of the Lrg21 bZip transcription factor gene during late differentiation. The synchronous expression of stage-specific and cell cycle genes regulated by E2a–Pbx1 in these cell lines comprises a model system in which analysis of their promoters can be used as a starting point to backtrack to the transcriptional mechanisms of oncogenesis by E2a–Pbx1.

Kinetics of CO and NO Ligation with the Cys331 → Ala Mutant of Neuronal Nitric-oxide Synthase

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX 4C) that participates in a ZnS4center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys331→ Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding ofl-Arg and tetrahydrobiopterin (BH4). Because binding l-Arg and BH4 to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH4 has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.

Rap1a deficiency modifies cytokine responses and MAPK-signaling in vitro and impairs the in vivo inflammatory response.

Rap1, which is closely related to ras, plays a key role in T-cell receptor (TCR)-signaling. TCR-stimulation without costimulation leads to constitutively activated rap1, which may mediate T-cell anergy via inhibition of ras-dependent induction of extracellular signal-regulated kinases (ERK). This activation is mediated by a second protein kinase b-Raf. Rap1-GTP is thought to activate ERK in a ras-independent manner by binding b-raf. Generally, T cells do not express b-raf while they express the adaptor protein raf-1, which is usually sequestered by rap1 leading to inhibition of ras-mediated ERK activation. In this study, we demonstrate that in rap1-deficient T cells, signaling by the ERK and p38 kinases is increased following activation by different stimuli leading to increased intracellular accumulation and secretion of cytokines. In addition, in a hypersensitivity model rap1-deficient mice demonstrated reduced contact dermatitis compared to wildtype mice, demonstrating the impact of rap1-deficiency on the inflammatory response in vivo.

Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity

BackgroundRetrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks.ResultsHere we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12th and 59th amino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio in vivo, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen.ConclusionsTogether, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitts lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS) warrants further investigation into its role in disease development.