BBA - Molecular Cell Research (v.1813, #2)

Control of nuclear HIPK2 localization and function by a SUMO interaction motif by Laureano de la Vega; Katrin Fröbius; Rita Moreno; Marco A. Calzado; Hui Geng; M. Lienhard Schmitz (283-297).
The serine/threonine kinase HIPK2 regulates gene expression programs controlling differentiation and cell death. HIPK2 localizes in subnuclear speckles, but the structural components allowing this localization are not understood. A point mutation analysis allowed mapping two nuclear localization signals and a SUMO interaction motif (SIM) that also occurs in HIPK1 and HIPK3. The SIM binds all three major isoforms of SUMO (SUMO-1–3), while only SUMO-1 is capable of covalent conjugation to HIPK2. Deletion or mutation of the SIM prevented SUMO binding and precluded localization of HIPK2 in nuclear speckles, thus causing localization of HIPK2 to the entire cell. Functional inactivation of the SIM prohibited recruitment of HIPK2 to PML nuclear bodies and disrupted colocalization with other proteins such as the polycomb protein Pc2 in nuclear speckles. Interaction of HIPK2 with Pc2 or PML in intact cells was largely dependent on a functional SIM in HIPK2, highlighting the relevance of SUMO/SIM interactions as a molecular glue that serves to enhance protein/protein interaction networks. HIPK2 mutants with an inactive SIM showed changed activities, thus revealing that non-covalent binding of SUMO to the kinase is important for the regulation of its function.►HIPK2 contains two nuclear localization sequences. ►The kinase contains a SUMO interaction motif, which is required for localization to nuclear speckles and recruitment to PML bodies. ►SUMO/SIM interactions control HIPK2 activity and protein/protein interactions.
Keywords: SIM; SUMO; HIPK2; Nuclear bodies; PML;

H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes by Jodi McKay; Xing Wang; Jian Ding; Janice E. Buss; Linda Ambrosio (298-307).
Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Instead, both oncogenic H-Ras-61L and wild type H-Ras (basal or EGF-stimulated) bind Arf6-associated clathrin-independent endosomes and vesicles of the endosomal-recycling center (ERC). K-Ras4B-12V can also be internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are sufficient to mediate localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6-associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the specificity of Ras:effector interaction.► Basal, EGF-stimulated and oncogenic H-Ras bind Arf6 endosomes in NIH3T3 cells. ► These forms of H-Ras are absent from endosomes of the clathrin-associated pathway. ► Very little Raf-1 is found associated with H-Ras on Arf6 vesicles. ► Instead Raf-1 distributes primarily to EEA-1 vesicles. ► Separation of H-Ras and Raf-1 during early endocytosis may impact signal output.
Keywords: Ras; Endocytosis; Arf6; Protein trafficking; Endosomes; Clathrin; Raf-1; Endosomal-recycling center;

Interleukin (IL)-6, -4, and -8 levels have been elevated in most patients suffering from prostate, breast, or colon cancer. There is a large body of evidence suggesting that chronic inflammation is one of the etiologic factors in these tumors. IL-6 is a multifunctional cytokine which is known to influence proliferation, apoptosis, and angiogenesis in cancer. Its transcription factor STAT3 is known as an oncogene that is constitutively phosphorylated in these malignancies. However, IL-6-induced STAT3 phosphorylation may result in growth arrest. IL-6 activation of androgen receptor in prostate cancer may yield either tumor cell proliferation or differentiation. Prolonged treatment with IL-6 results in generation of sublines which express a more malignant phenotype. Therapy options against IL-6 have been established and the antibody siltuximab has been applied in preclinical and clinical studies. Recently, investigations of the role of suppressors of cytokine signaling have been carried out. IL-4 and -8 are implicated in regulation of apoptosis, migration, and angiogenesis in cancers associated with chronic inflammation. All cytokines mentioned above regulate cellular events in stem cells. These cells could not be targeted by most conventional cancer therapies.► Interleukin-6 has multiple roles in inflammation-associated cancers. ► Signaling pathways of STAT are constitutively active in common human cancers. ► Therapy with an interleukin-6 antibody has been established in cancer models. ► Interleukin-4 and -8 promote migration, angiogenesis, and metastasis.
Keywords: Interleukin; Prostate cancer; Breast cancer; Colon cancer; Apoptosis; Angiogenesis;

Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells by Drew Williams; Grant Norman; Chamel Khoury; Naomi Metcalfe; Jennie Briard; Aimee Laporte; Sara Sheibani; Liam Portt; Craig A. Mandato; Michael T. Greenwood (315-321).
The identification of novel anti-apoptotic sequences has lead to new insights into the mechanisms involved in regulating different forms of programmed cell death. For example, the anti-apoptotic function of free radical scavenging proteins supports the pro-apoptotic function of Reactive Oxygen Species (ROS). Using yeast as a model of eukaryotic mitochondrial apoptosis, we show that a cDNA corresponding to the mitochondrial variant of the human DUT gene (DUT-M) encoding the deoxyuridine triphosphatase (dUTPase) enzyme can prevent apoptosis in yeast in response to internal (Bax expression) and to exogenous (H2O2 and cadmium) stresses. Of interest, cell death was not prevented under culture conditions modeling chronological aging, suggesting that DUT-M only protects dividing cells. The anti-apoptotic function of DUT-M was confirmed by demonstrating that an increase in dUTPase protein levels is sufficient to confer increased resistance to H2O2 in cultured C2C12 mouse skeletal myoblasts. Given that the function of dUTPase is to decrease the levels of dUTP, our results strongly support an emerging role for dUTP as a pro-apoptotic second messenger in the same vein as ROS and ceramide.► dUTPase is capable of preventing Bax induced cell death in yeast. ► dUTPase prevents oxidative stress mediated cell death in yeast. ► dUTPase prevents oxidative stress mediated cell death in cultured mammalian cells. ► The anti-apoptotic function of dUTPase suggests that dUTP is a pro-apoptotic second messenger that serves to mediate the effects of stress on cell death.
Keywords: Apoptosis; Cell death; Anti-death; Anti-apoptosis; Anti-apoptotic; Cell survival; Yeast; C2C12 cells; dUTP;

eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1 by Brynhildur Thors; Haraldur Halldórsson; Gudmundur Thorgeirsson (322-331).
Reports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca+ 2/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and − α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK.► G-proteins agonists cause eNOS phosphorylation when AMPK is activated by LKB1. ► AMPK phosphorylates eNOS when intracellular ATP is lowered. ► AMPKα2 has a role in maintaining endothelial monolayer integrity.
Keywords: eNOS; AMPK; Histamine; LKB1; Endothelial cells;

The Type III TGF-β receptor, betaglycan, is a widely expressed proteoglycan co-receptor for TGF-β superfamily ligands. The full-length protein undergoes ectodomain cleavage with release of a soluble ectodomain fragment. The fate of the resulting transmembrane–cytoplasmic fragment, however, has never been explored. We demonstrate here that the transmembrane–cytoplasmic fragment is stable in transfected cells and in cell lines expressing endogenous betaglycan. Production of this fragment is inhibited by the ectodomain shedding inhibitor TAPI-2. Treatment of cells with inhibitors of the intramembrane protease γ-secretase stabilizes this fragment, suggesting that it is a substrate of γ-secretase. Expression of the transmembrane–cytoplasmic fragment as well as γ-secretase inhibitor stabilization are independent of TGF-β1 or -β2 and are unaffected by mutation of the cytoplasmic domain serines that undergo phosphorylation. γ-Secretase inhibition or the expression of a transmembrane–cytoplasmic fragment in HepG2 cells blunted TGF-β2 signaling. Our findings thus suggest that the transmembrane–cytoplasmic fragment remaining after betaglycan ectodomain cleavage is stable and a substrate of γ-secretase, which may have significant implications for the TGF-β signaling response.► Betaglycan ectodomain shedding yields a stable transmembrane–cytoplasmic fragment. ► The transmembrane–cytoplasmic fragment is a target of γ-secretase. ► γ-Secretase inhibitors suppress TGF-β2-related signaling.
Keywords: TGF-β; Betaglycan; γ-Secretase; Ectodomain shedding; Regulated intramembrane proteolysis;

Estradiol stabilizes the 105-kDa phospho-form of the adhesion docking protein NEDD9 and suppresses NEDD9-dependent cell spreading in breast cancer cells by Lauren N. Bradshaw; J. Zhong; P. Bradbury; Maha Mahmassani; Jessica L. Smith; Alaina J. Ammit; Geraldine M. O'Neill (340-345).
Recent data suggest that the adhesion docking protein NEDD9/HEF1/Cas-L is a critical regulator of adhesion-dependent signalling pathways during mammary tumour development. Multiple phosphorylation modifications of NEDD9 regulate interaction with downstream protein partners, thus the regulation of NEDD9 phospho-forms is an important point of control for NEDD9 function. As estradiol (E2) plays a central role in the development and progression of breast cancer, we have investigated NEDD9 phospho-form regulation in MCF-7 estrogen receptor (ER)-positive breast cancer cells in response to estrogen. We find that levels of the 105-kDa NEDD9 phospho-form are significantly increased after 3 days of estrogen exposure, and this is suppressed by the anti-estrogen tamoxifen. Analysis of protein decay kinetics following treatment with the protein synthesis inhibitor cycloheximide indicates that increased 105-kDa levels are due to a slower rate of protein decay. Moreover, exogenous expression of NEDD9 failed to induce spreading in the presence of E2, and this was reversed by tamoxifen treatment. Finally, we show that the 105-kDa NEDD9 phospho-form appears to predominate in ER-positive versus ER-negative breast cancer cell lines. Taken together, our results suggest that estradiol may suppress phospho-form-specific functions of NEDD9.► Estradiol regulates the stability of NEDD9 phospho-forms. ► NEDD9-mediated cell spreading is inhibited by estradiol. ► Estradiol suppresses phospho-form-specific functions of NEDD9.
Keywords: Breast cancer; Estrogen; NEDD9; Phosphorylation;

Modulation of α2C adrenergic receptor temperature-sensitive trafficking by HSP90 by Catalin M. Filipeanu; René de Vries; A.H. Jan Danser; Daniel R. Kapusta (346-357).
Decreasing the temperature to 30 °C is accompanied by significant enhancement of α2C-AR plasma membrane levels in several cell lines with fibroblast phenotype, as demonstrated by radioligand binding in intact cells. No changes were observed on the effects of low-temperature after blocking receptor internalization in α2C-AR transfected HEK293T cells. In contrast, two pharmacological chaperones, dimethyl sulfoxide and glycerol, increased the cell surface receptor levels at 37 °C, but not at 30 °C. Further, at 37 °C α2C-AR is co-localized with endoplasmic reticulum markers, but not with the lysosomal markers. Treatment with three distinct HSP90 inhibitors, radicicol, macbecin and 17-DMAG significantly enhanced α2C-AR cell surface levels at 37 °C, but these inhibitors had no effect at 30 °C. Similar results were obtained after decreasing the HSP90 cellular levels using specific siRNA. Co-immunoprecipitation experiments demonstrated that α2C-AR interacts with HSP90 and this interaction is decreased at 30 °C. The contractile response to endogenous α2C-AR stimulation in rat tail artery was also enhanced at reduced temperature. Similar to HEK293T cells, HSP90 inhibition increased the α2C-AR contractile effects only at 37 °C. Moreover, exposure to low-temperature of vascular smooth muscle cells from rat tail artery decreased the cellular levels of HSP90, but did not change HSP70 levels. These data demonstrate that exposure to low-temperature augments the α2C-AR transport to the plasma membrane by releasing the inhibitory activity of HSP90 on the receptor traffic, findings which may have clinical relevance for the diagnostic and treatment of Raynaud Phenomenon.► At 37 °C α2C-AR is localized mostly in the endoplasmic reticulum in HEK293T cells. ► Exposure to low-temperature stimulated the receptor transport to the cell surface. ► Inhibition of HSP90 has similar effects on the α2C-AR traffic as low-temperature. ► In rat tail artery, HSP90 inhibitors increase the contraction to α2C-AR stimulation. ► These findings may have clinical relevance in Raynaud Phenomenon.
Keywords: α2C-AR; HSP90; Heat shock proteins; Intracellular traffic; Molecular chaperones; Low temperature; Raynaud Phenomenon;

Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p by Kristofer Bodvard; David Wrangborg; Sofia Tapani; Katarina Logg; Piotr Sliwa; Anders Blomberg; Mats Kvarnström; Mikael Käll (358-366).
Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters. We here investigate how continuous illumination with blue light, corresponding to GFP excitation wavelengths, affects the localization pattern of Msn2p-GFP in budding yeast. The localization pattern was analyzed using a novel approach that combines wavelet decomposition and change point analysis. It was found that the Msn2p nucleocytoplasmic localization trajectories for individual cells exhibit up to three distinct and successive states; i) Msn2p localizes to the cytoplasm; ii) Msn2p rapidly shuttles between the cytoplasm and the nucleus; iii) Msn2p localizes to the nucleus. Many cells pass through all states consecutively at high light intensities, while at lower light intensities most cells only reach states i) or ii). This behaviour strongly indicates that continuous light exposure gradually increases the stress level over time, presumably through continuous accumulation of toxic photoproducts, thereby forcing the cell through a bistable region corresponding to nucleocytoplasmic oscillations. We also show that the localization patterns are dependent on protein kinase A (PKA) activity, i.e. yeast cells with constantly low PKA activity showed a stronger stress response. In particular, the nucleocytoplasmic oscillation frequency was found to be significantly higher for cells with low PKA activity for all light intensities.► Continuous illumination causes Msn2p to exhibit three distinct localization states. ► At higher light intensities cells are more likely to exhibit all three Msn2p states. ► Low PKA activity increases Msn2p oscillation frequency. ► Continuous illumination cause increasing stress levels.
Keywords: Light stress; Transcription factor; Nuclear oscillations; Fluorescence microscopy; Yeast; Msn2p;

The effects of membrane compartmentalization of csk on TCR signaling by Pavel Otáhal; Supansa Pata; Pavla Angelisová; Václav Hořejší; Tomáš Brdička (367-376).
The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.► Csk targeted to lipid rafts inhibits TCR signaling. ► Membrane raft-associated Src-family kinases play critical role in TCR signal transduction. ► Functional evidence for in vivo importance of membrane rafts.
Keywords: Lipid rafts; T-cell receptor; Lck; Csk;