Stephan A. Eisler

Global Detection of Protein Kinase D-dependent Phosphorylation Events in Nocodazole-treated Human Cells

Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells.

A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.

The protein kinase D (PKD) family comprises multifunctional serine/threonine‐specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral‐targeted transport carrier fission from the trans‐Golgi network (TGN). To visualize site‐specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD‐specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi‐specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD‐specific pharmacological inhibitors and siRNA‐mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole‐induced Golgi complex break‐up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant‐negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.

The cofilin phosphatase slingshot homolog 1 (SSH1) links NOD1 signaling to actin remodeling.

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-κB 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-κB activation and innate immune responses.

The Rho-specific GAP protein DLC3 coordinates endocytic membrane trafficking

ABSTRACT 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.

PKD controls mitotic Golgi complex fragmentation through a Raf-MEK1 pathway.

Protein kinase D (PKD) is known to be involved in the fission of transport carriers at the Golgi complex. This study demonstrates that PKD is important for the cleavage of interstack Golgi connections in G2 of the cell cycle and thus entry of cells into mitosis.

The tumor suppressor protein DLC1 is regulated by PKD-mediated GAP domain phosphorylation

Deleted in liver cancer 1 (DLC1) is a tumor suppressor protein that is frequently downregulated in various tumor types. DLC1 contains a Rho GTPase activating protein (GAP) domain that appears to be required for its tumor suppressive functions. Little is known about the molecular mechanisms that regulate DLC1. By mass spectrometry we have mapped a novel phosphorylation site within the DLC1 GAP domain on serine 807. Using a phospho-S807-specific antibody, our results identify protein kinase D (PKD) to phosphorylate this site in DLC1 in intact cells. Although phosphorylation on serine 807 did not directly impact on in vitro GAP activity, a DLC1 serine-to-alanine exchange mutant inhibited colony formation more potently than the wild type protein. Our results thus show that PKD-mediated phosphorylation of DLC1 on serine 807 negatively regulates DLC1 cellular function.

G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi-network.

The serine/threonine protein kinase D (PKD) is recruited to the trans‐Golgi‐network (TGN) by interaction with diacylglycerol (DAG) and Arf1 and promotes the fission of vesicles containing cargo destined for the plasma membrane. PKD activation is mediated by PKC(‐induced phosphorylation. However, signaling pathways that activate PKD specifically at the TGN are only poorly characterized. Recently we created G‐PKDrep, a genetically encoded fluorescent reporter for PKD activity at the TGN in fixed cells. To establish a reporter useful for monitoring Golgi‐specific PKD activity in living cells we now refined G‐PKDrep to generate G‐PKDrep‐live. Specifically, phosphorylation of G‐PKDrep‐live expressed in mammalian cells results in changes of fluorescence resonance energy transfer (FRET), and allows for indirect imaging of PKD activity. In a proof‐of‐principle experiment using phorbolester treatment, we demonstrate the reporters capability to track rapid activation of PKD at the TGN. Furthermore, activation‐induced FRET changes are reversed by treatment with PKD‐specific pharmacological inhibitors. Thus, the newly developed reporter G‐PKDrep‐live is a suitable tool to visualize dynamic changes in PKD activity at the TGN in living cells.

Novel strategies to mimic transmembrane tumor necrosis factor-dependent activation of tumor necrosis factor receptor 2

Tumor necrosis factor receptor 2 (TNFR2) is known to mediate immune suppression and tissue regeneration. Interestingly, the transmembrane form of tumor necrosis factor (tmTNF) is necessary to robustly activate TNFR2. To characterize the stoichiometry and composition of tmTNF during TNFR2 activation, we constructed differently oligomerized single chain TNF ligands (scTNF) comprised of three TNF homology domain (THD) protomers that mimic tmTNF. Using a variety of cellular and in vivo assays, we can show that higher oligomerization of the scTNF trimers results in more efficient TNF/TNFR2 clustering and subsequent signal transduction. Importantly, the three-dimensional orientation of the scTNF trimers impacts the bioactivity of the oligomerized scTNF ligands. Our data unravel the organization of tmTNF-mimetic scTNF ligands capable of robustly activating TNFR2 and introduce novel TNFR2 agonists that hold promise as therapeutics to treat a variety of diseases.

A Rho signaling network links microtubules to PKD controlled carrier transport to focal adhesions

Protein kinase D (PKD) is a family of serine/threonine kinases that is required for the structural integrity and function of the Golgi complex. Despite its importance in the regulation of Golgi function, the molecular mechanisms regulating PKD activity are still incompletely understood. Using the genetically encoded PKD activity reporter G-PKDrep we now uncover a Rho signaling network comprising GEF-H1, the RhoGAP DLC3, and the Rho effector PLCε that regulate the activation of PKD at trans-Golgi membranes. We further show that this molecular network coordinates the formation of TGN-derived Rab6-positive transport carriers delivering cargo for localized exocytosis at focal adhesions.

FRET-Biosensoren: molekulare Werkzeuge in der Signalwegforschung

Genetically encoded, FRET based biosensors have revolutionized cell signalling research in the past decade. They allow tracking dynamic activities of signalling molecules such as protein kinases, Rho GTPases or second messengers in real time on a subcellular level. This article gives an overview about design, components and employment of different types of FRET biosensors. Exemplarily, a kinase activity reporter to visualize protein kinase D activity at the Golgi complex is presented.