Angelika Hausser - Academia.edu (original) (raw)

Papers by Angelika Hausser

e here describe the structural requirements for Golgi localization and a sequential, localization... more e here describe the structural requirements for Golgi localization and a sequential, localizationdependent activation process of protein kinase C (PKC) involving auto-and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC -green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH 2 -terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC completely abrogated Golgi localization of PKC . As an NH 2 -terminal PKC fragment was colocalized with p24, this region of PKC is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed W the constitutive, rapid recruitment of cytosolic PKC to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinasedead PKC found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC , in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto-and transphosphorylation of NH 2 -terminal serine(s) in the regulatory domain. PKC activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC function at the Golgi compartment.

The Journal of cell biology, Jan 7, 2002

We here describe the structural requirements for Golgi localization and a sequential, localizatio... more We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC) mu involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC mu-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH(2)-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC mu completely abrogated Golgi localization of PKC mu. As an NH(2)-terminal PKC mu fragment was colocalized with p24, this region of PKC mu is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC mu to, and stable association with, the Golgi compartment independent of activation loop ph...

Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2015

Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons.... more Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons. During the search for future presynaptic partners, their shape and size change dynamically, with a direct impact on the formation, stabilization and maintenance of synaptic connections both in vivo and in vitro. In order to reveal molecular players regulating synapse formation, quantitative analysis of dendritic filopodia motility is needed. Defining the length or the tips of these protrusions manually, however, is time consuming, limiting the extent of studies as well as their statistical power. Additionally, area detection based on defining a single intensity threshold can lead to significant errors throughout the image series, as these small structures often have low contrast in fluorescent images. To overcome these problems, the open access Dendritic Filopodia Motility Analyzer, a semi-automated ImageJ/Fiji plugin was created. Our method calculates the displacement of the centre of ...

Journal of Biotechnology, 2009

Recent studies have demonstrated that the introduction of transgenes regulating protein transport... more Recent studies have demonstrated that the introduction of transgenes regulating protein transport or affecting post-translational modifications can further improve industrial processes for the production of therapeutic proteins in mammalian cells. Our study on improving therapeutic protein production in CHO cells by heterologous expression of the ceramide transfer protein (CERT) was initiated by the recent discovery that CERT is involved in

Journal of Cell Science, 2015

Membrane trafficking is known to be coordinated by small GTPases, but the identity of their regul... more Membrane trafficking is known to be coordinated by small GTPases, but the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure balanced GTPase activation at different subcellular sites is largely elusive. Here, we show in living cells that deleted in liver cancer 3 (DLC3, also known as STARD8) is a functional Rho-specific GAP protein, the loss of which enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and is required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment (ERC), which is restored by the simultaneous downregulation of RhoA and RhoB. We further demonstrate that DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation associated with prolonged receptor signaling. Taken together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport through the control of Rho activity.

PLoS Pathogens, 2014

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate ... more NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-kB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-kB activation and innate immune responses.

Traffic, 2009

† Both authors contributed equally to this work.

The Journal of Cell Biology, 2007

Abbreviations used in this paper: KD, kinase dead; MLV, multilamellar vesicle; PC, phosphatidylch... more Abbreviations used in this paper: KD, kinase dead; MLV, multilamellar vesicle; PC, phosphatidylcholine; PH, pleckstrin homology; PI(4)P, phosphatidylinositol 4phosphate; SM, sphingomyelin; START, steroidogenic acute regulatory lipid transfer; TNP-PE, 2,4,6-trinitrophenyl-phosphatidylethanolamine; WT, wild type.

The International Journal of Biochemistry & Cell Biology, 2010

The adapter protein SLy2 (SH3 protein expressed in lymphocytes 2), also named HACS1, NASH1 or SAM... more The adapter protein SLy2 (SH3 protein expressed in lymphocytes 2), also named HACS1, NASH1 or SAMSN1, is expressed in hematopoietic tissues, muscle, heart, brain, lung, pancreas, endothelial cells and myelomas. Endogenous SLy2 expression was shown to be upregulated in primary B cells upon differentiation and proliferation-inducing stimuli, and transduction experiments suggest a stimulatory role for SLy2 in B cell differentiation to plasma cells. However the signalling pathways regulated by SLy2 remain unknown. In this study we identify novel interaction partners of SLy2 providing a molecular framework for its function. We show that phosphorylated SLy2 directly interacts with 14-3-3 proteins via a previously unrecognized phosphorylation site. Furthermore, we demonstrate that 14-3-3 proteins control nucleo-cytoplasmic shuttling of SLy2 by retaining phosphorylated SLy2 in the cytoplasm. In the nucleus, SLy2 interacts with the SAP30/HDAC1 complex and regulates the activity of HDAC1. Thus, our findings unravel a novel mechanism how SLy2 localization is controlled and implicate SLy2 in the epigenetic control of gene expression.

Stem Cells and Development, 2014

Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells,... more Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, which are able to differentiate in vitro into osteocytes, adipocytes, and chondrocytes. Mouse BMSCs (mBMSCs) are a versatile model system to investigate factors involved in BMSC differentiation in vitro and in vivo as a variety of transgenic mouse models are available. In this study, mBMSCs were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy.

Nature Cell Biology, 2005

Protein kinase D (PKD) regulates the fission of vesicles from the trans-Golgi-network 1,2 . We sh... more Protein kinase D (PKD) regulates the fission of vesicles from the trans-Golgi-network 1,2 . We show that phosphatidylinositol-4-kinase III beta (PI4KIIIβ), a key player in Golgi complex structure and function 3 , is a physiological substrate of PKD. Of the three PKD isoforms, only PKD1 and PKD2 phosphorylated PI4KIIIβ at a motif highly conserved from yeast to man. PKD mediated phosphorylation stimulated lipid kinase activity of PI4KIIIβ and enhanced VSV-G transport to plasma membrane. The identification of PI4KIIIβ as one of the PKD substrates should help to reveal the molecular events leading to transport carrier formation.

Journal of Cell Science, 2006

Journal of Cell Science, 2009

Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated i... more Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated in various tumor types. In vitro, DLC1 specifically inactivates the small GTPases RhoA, RhoB and RhoC through its GAP domain and this appears to contribute to its tumor suppressor function in vivo. Molecular mechanisms that control DLC1 activity have not so far been investigated. Here, we show that phorbol-ester-induced activation of protein kinase C and protein kinase D stimulates association of DLC1 with the phosphoserine/phosphothreonine-binding 14-3-3 adaptor proteins via recognition motifs that involve Ser327 and Ser431. Association with 14-3-3 proteins inhibits DLC1 GAP activity and facilitates signaling by active Rho. We further show that treatment of cells with phorbol ester or coexpression of 14-3-3 proteins, blocks DLC1 nucleocytoplasmic shuttling, probably by masking a previously unrecognized nuclear localization sequence. The binding to 14-3-3 proteins is thus a newly discovered mechanism by which DLC1 activity is regulated and compartmentalized.

Journal of Biological Chemistry, 2010

We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein c... more We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility.

Journal of Biological Chemistry, 1999

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in si... more Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3 isoform has been shown to associate with protein kinase C and to negatively regulate interleukin-2 secretion. Here we present data that

Journal of Biological Chemistry, 2008

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. ... more Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase C␥-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

$Research paper thumbnail of Drosophila protein kinase D is broadly expressed and a fraction localizes to the Golgi compartment$

Gene Expression Patterns, 2006

Protein kinase D belongs to the subfamily of CaMK. In mammals, three isoforms are known. They hav... more Protein kinase D belongs to the subfamily of CaMK. In mammals, three isoforms are known. They have been linked to diverse cellular functions including regulation of cell proliferation, differentiation, apoptosis and motility as well as secretory transport from the trans-Golgi compartment to the plasma membrane. Accordingly, the mammalian PKDs show different intracellular locations, with reported dynamic redistribution, between cytosol, Golgi, plasma membranes and the nucleus, depending on the cell type and exogenous stimuli. The genome of Drosophila melanogaster harbours just one, highly conserved PKD homologue, which is expressed throughout development. PKD mRNA expression during late embryogenesis is restricted to ectodermal derivatives including those involved in cuticle secretion. In imaginal tissues, transcription appears more uniform. PKD protein is detected predominantly in the cytosol with an enrichment in lateral apodemes of late embryos as well as in larval fascicles. In secretory tissues like salivary glands, the protein is concentrated in dotted structures. A PKD-GFP transgene reveals a similar punctuate protein accumulation juxtaposed to a resident Golgi-marker. In cultured cells, transfected Drosophila PKD-GFP colocalizes with a marker of the trans-Golgi compartment like human PKD1-GFP. Similar to the mammalian homologues, Drosophila PKD may be multifunctional including a role in secretory transport in accordance with its subcellular distribution.

Frontiers in Systems Neuroscience, 2009

Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD i... more Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1 S738/742E (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1 K612W (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.

FEBS Letters, 2007

Protein kinase D (PKD) has been implicated in the regulation of cell shape, adhesion, and migrati... more Protein kinase D (PKD) has been implicated in the regulation of cell shape, adhesion, and migration. At the leading edge of migrating cells active PKD co-localizes with F-actin, Arp3 and cortactin. Platelet derived growth factor (PDGF) activates PKD and recruits the kinase to the leading edge, suggesting a role for PKD in actin remodelling. In support of this, PKD directly interacts with F-actin and phosphorylates cortactin in vitro. Interference with PKD function by overexpression of a dominant negative PKD or by PKD-specific siRNA enhanced cell migration, whereas cells overexpressing PKD wild type displayed reduced migratory potential. Taken together, these data reveal a negative regulatory function of PKD in cell migration.

FEBS Letters, 1999

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutationa... more Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, Xchromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCW W, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of Bcells with phorbol ester or H 2 O 2 affected Btk/PKCW W interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCW W. Transient overexpression of PKCW W deletion mutants as well as expression of selected PKCW W domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCW W/Btk complex in vivo and identify two PKCW W domains that participate in Btk interaction.