Dirk Fahrenkamp

Src family kinases mediate cytoplasmic retention of activated STAT5 in BCR–ABL-positive cells

Persistent activation of the Abl tyrosine kinase in the BCR–ABL fusion protein is the major cause of chronic myeloid leukemia (CML). Among many other substrates BCR–ABL phosphorylates STAT5 and Src family kinases (SFK). Activated pSTAT5 is essential for initial transformation and maintenance of the disease. Cytokine-induced phosphorylation on tyrosine 694 typically leads to nuclear accumulation of pSTAT5 and target gene expression. We verified that in BCR–ABL-positive progenitor cells from a CML patient and in K562 cells pSTAT5 is cytoplasmic. However, upon ectopic expression of BCR–ABL p210 in non-myeloid cells, co-transfected STAT5A is phosphorylated on Y694 and localized in the nucleus arguing for an additional factor mediating cytoplasmic retention in CML cells. Expression of the SFK v-Src, Hck or Lyn together with STAT5A results in phosphorylation on Y694 and cytoplasmic retention. Upon coexpression of BCR–ABL and individual SFK the cytoplasmic retention of activated STAT5A mediated by v-Src and Hck but not Lyn is dominant over nuclear translocation induced by BCR–ABL. Cytoplasmic retention depends on the kinase activity of SFK and is mediated through the interaction of the SH2 domain of STAT5A with the SFK. Interestingly, nuclear accumulation of STAT5A as a result of activation by FLT3-ITD, an oncogene found in acute myeloid leukemia, cannot be prevented by coexpression of SFK. Importantly, inhibition of SFK in K562 cells restored nuclear accumulation of pSTAT5A, enhanced STAT5 target gene expression and increased colony formation. Thus, SFK mediate cytoplasmic retention of pSTAT5A in BCR–ABL-positive cells. Cytoplasmic pSTAT5A in CML cells might balance the controversial functions of STAT5 in cellular senescence and differentiation versus G1/S progression and survival.

Activated fibronectin-secretory phenotype of mesenchymal stromal cells in pre-fibrotic myeloproliferative neoplasms

We characterized bone marrow stromal cells (BMSC) from patients with pre-fibrotic myeloproliferative neoplasms (MPN). MPN-BMSC showed decreased capacity to stimulate the proliferation of colony-forming units of normal hematopoietic stem and progenitor cells and displayed increased matrix remodelling (in particular fibronectin deposition) compared to control BMSC. This finding was confirmed in pre-fibrotic MPN bone marrow biopsies in a tissue microarray (n = 34), which stained positive for fibronectin in the absence of reticulin as a standard myelofibrosis marker. Fibronectin expression correlated significantly with reduced haemoglobin levels in MPN-patients (p = 0.007; R2 = 0.42). Our data show significant cell-intrinsic alterations in MPN-MSC and suggest that Fibronectin expression might be applicable as a biomarker for the identification of early myelofibrotic transformation in reticulin-negative MPN.

Consequences of the disease-related L78R mutation for dimerization and activity of STAT3

ABSTRACT Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is centrally involved in diverse processes including haematopoiesis, immunity and cancer progression. In response to cytokine stimulation, STAT3 is activated through phosphorylation of a single tyrosine residue. The phosphorylated STAT3 dimers are stabilized by intermolecular interactions between SH2 domains and phosphotyrosine. These activated dimers accumulate in the nucleus and bind to specific DNA sequences, resulting in target gene expression. We analysed and compared the structural organizations of the unphosphorylated latent and phosphorylated activated STAT3 dimers using Förster resonance energy transfer (FRET) in fixed and living cells. The latent dimers are stabilized by homotypic interactions between the N-terminal domains. A somatic mutation (L78R) found in inflammatory hepatocellular adenoma (IHCA), which is located in the N-terminal domain of STAT3 disturbs latent dimer formation. Applying intramolecular FRET, we verify a functional role of the SH2 domain in latent dimer formation suggesting that the protomers in the latent STAT3 dimer are in a parallel orientation, similar to activated STAT3 dimers but different from the antiparallel orientation of the latent dimers of STAT1 and STAT5. Our findings reveal unique structural characteristics of STAT3 within the STAT family and contribute to the understanding of the L78R mutation found in IHCA.

Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration.

Dominant-negative activity of the STAT3-Y705F mutant depends on the N-terminal domain

BackgroundSTAT3 is a transcription factor of central importance in chronic inflammation and cancer. In response to cytokine stimulation STAT3 is phosphorylated on a single tyrosine residue at position 705, dimerizes and accumulates in the nucleus to induce target gene expression. The substitution of tyrosine 705 to phenylalanine leads to a dominant-negative STAT3 mutant (STAT3-YF) which influences the activation of WT-STAT3 in stimulated cells through a mechanism that is not completely understood. In this study we analyzed the molecular mechanism of STAT3-YF dominant-negative activity in IL-6-induced STAT3 signaling and the relevance of the N-terminal domain.ResultsExpression of STAT3-YF-YFP impairs tyrosine phosphorylation, nuclear translocation and the transcriptional activity of WT-STAT3 in IL-6-stimulated cells. The fluorescently labelled STAT3-YF mutant binds to a phosphorylated gp130 receptor-peptide comparable to WT-STAT3-YFP. STAT3-YF-YFP forms homodimers as well as heterodimers with WT-STAT3 in the presence and absence of IL-6. The preformed heterodimers in unstimulated cells are detectable by colocalization of STAT3-CFP with STAT3-YF-YFP fused to a nuclear localization signal. STAT3/STAT3-YF heterodimers are not able to bind to DNA in stimulated cells, but the presence of the mutant reduces DNA-binding of WT-STAT3 homodimers. STAT3-YF-ΔN-YFP lacking the N-terminal domain forms no dimers and only marginally affects the activity of WT-STAT3.ConclusionOur findings demonstrate that dominant-negative STAT3-YF affects the activation of WT-STAT3 at multiple levels. Unexpectedly, the N-terminal domain of STAT3-YF plays an important role for the dominant-negative effect. We show that (i) STAT3-YF competes with WT-STAT3 in binding to activated gp130-receptors, (ii) the formation of WT-STAT3/STAT3-YF heterodimers in IL-6-stimulated cells results in inactive, semiphosphorylated dimers which do not bind to DNA and thus fail to induce target gene expression, (iii) the N-terminal domain-mediated formation of preformed STAT3/STAT3-YF heterodimers in unstimulated cells which affects the IL-6-induced homodimerization of WT-STAT3 contributes to the dominant-negative effect of STAT3-YF. These findings will contribute to our understanding of naturally occuring dominant-negative STAT3 mutants that cause the hyper-IgE syndrome.

Intramolecular hydrophobic interactions are critical mediators of STAT5 dimerization

STAT5 is an essential transcription factor in hematopoiesis, which is activated through tyrosine phosphorylation in response to cytokine stimulation. Constitutive activation of STAT5 is a hallmark of myeloid and lymphoblastic leukemia. Using homology modeling and molecular dynamics simulations, a model of the STAT5 phosphotyrosine-SH2 domain interface was generated providing first structural information on the activated STAT5 dimer including a sequence, for which no structural information is available for any of the STAT proteins. We identified a novel intramolecular interaction mediated through F706, adjacent to the phosphotyrosine motif, and a unique hydrophobic interface on the surface of the SH2 domain. Analysis of corresponding STAT5 mutants revealed that this interaction is dispensable for Epo receptor-mediated phosphorylation of STAT5 but essential for dimer formation and subsequent nuclear accumulation. Moreover, the herein presented model clarifies molecular mechanisms of recently discovered leukemic STAT5 mutants and will help to guide future drug development.

Assessing the Intracellular Integrity of Phosphine‐Stabilized Ultrasmall Cytotoxic Gold Nanoparticles Enabled by Fluorescence Labeling

As the size of nanoparticles (NPs) is in the range of biological molecules and subcellular structures, they provide new perspectives in biomedicine. This work presents studies concerning the cellular uptake and distribution of phosphine-stabilized cytotoxic 1.4 nm sized AuNPs and their probable degradation during this process. Therefore, ultrasmall phosphine-stabilized AuNPs are modified by linking a fluorophore covalently to the ligand shell. Monitoring the fluorescence on a cellular level by means of flow cytometry and confocal laser scanning microscopy allows determining the fate of the ligand shell during AuNP cell internalization, due to the fact that the fluorescence of a fluorophore bound near to the AuNP surface is quenched. Cell fractionation is conducted in order to quantify the AuNP content at the cell membrane, in the cytoplasm, and the cell nucleus. The incubation of cells with the fluorophore-modified AuNPs reveals a partial loss of the ligand shell upon AuNP cell interaction, evident by the emerging fluorescence signal. This loss is the precondition to unfold high AuNP cytotoxicity. Together with their significantly different biodistribution and enhanced circulation times compared to larger AuNPs, the findings demonstrate the high potential of ultrasmall AuNPs for drug development or therapy.

Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking.

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the subcellular localization of this mutant had not changed. Our findings support the concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition.

Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

BackgroundChronic myeloid leukemia (CML) is driven by the expression of the BCR-ABL oncoprotein. STAT5 is a BCR-ABL substrate and persistently activated by tyrosine phosphorylation in CML cells. Activated STAT5 (pSTAT5) drives proliferation and survival of leukemic cells and contributes to initial transformation and maintenance of the disease. In cytokine-induced STAT5 signaling, phosphorylation of STAT5A on Y694 leads to nuclear accumulation of the transcription factor, followed by DNA-binding and gene induction. However, Src-family kinases (SFK) mediate cytoplasmic retention of pSTAT5A leading to attenuated target gene expression and colony formation in CML cells.ResultsIn this study we show that autophosphorylation of Y416 in the highly conserved activation loop of SFK generates a potent recruitment site for the SH2 domain of STAT5A. Binding of the SH2 domain to the activation loop is required for STAT5AY694 phosphorylation by SFK, but at the same time promotes the persistent cytoplasmic localization of the transcription factor as found in BCR-ABL+ leukemia. As a consequence of the complex formation between tyrosine-phosphorylated SFK and the SH2 domain of STAT5A, the dimerization of STAT5A is impaired. We further demonstrate that constitutively active STAT5AS710F escapes from SFK-mediated cytoplasmic retention by enhancing STAT5A dimer stability.ConclusionOur results reveal important structural aspects of cytoplasmic pSTAT5A found in myeloid leukemias and will contribute to the understanding of STAT5A mediated cytoplasmic signaling.

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma

Significance The two E-type cyclins E1 and E2 are known to interact with Cdk2 and are thought to trigger cell cycle activity in carcinogenesis. However, the individual contributions of cyclin E1, cyclin E2, and Cdk2 for initiation and progression of hepatocellular carcinoma (HCC) are unknown. In the present study, we discovered that only cyclin E1—but not cyclin E2—is essential for initiation of liver cancer and requires Cdk2. Unexpectedly, advanced liver cancer progression can be mediated in presence of any E-cyclin, but in a Cdk2-independent manner. We identified the specific expression profiles of cyclin E1-dependent and cyclin E1-independent hepatoma cells. These signatures are useful for predicting patient prognosis and for developing novel cyclin E-based HCC therapies. E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.