PKD autoinhibition in trans regulates activation loop autophosphorylation in cis.

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation.

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.

DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.

The Rho-specific GAP protein DLC3 coordinates endocytic membrane trafficking.

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.

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.

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.

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.

Optimization of Mechanosensitive Cross-Talk between Matrix Stiffness and Protein Density: Independent Matrix Properties Regulate Spreading Dynamics of Myocytes.

Cells actively sense differences in topology, matrix elasticity and protein composition of the extracellular microenvironment and adapt their function and morphology. In this study, we focus on the cross-talk between matrix stiffness and protein coating density that regulates morphology and proliferation dynamics of single myocytes. For this, C2C12 myocytes were monitored on L-DOPA functionalized hydrogels of 22 different elasticity and fibronectin density compositions. Static images were recorded and statistically analyzed to determine morphological differences and to identify the optimized extracellular matrix (ECM). Using that information, selected ECMs were used to study the dynamics before and after cell proliferation by statistical comparison of distinct cell states. We observed a fibronectin-density-independent increase of the projected cell area until 12 kPa. Additionally, changes in fibronectin density led to an area that was optimum at about 2.6 µg/cm2, which was confirmed by independent F-actin analysis, revealing a maximum actin-filament-to-cell-area ratio of 7.5%. Proliferation evaluation showed an opposite correlation between cell spreading duration and speed to matrix elasticity and protein density, which did not affect cell-cycle duration. In summary, we identified an optimized ECM composition and found that independent matrix properties regulate distinct cell characteristics.

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.

Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development.

Epithelial ovarian cancer (EOC) is the most lethal disease of the female reproductive tract, and although most patients respond to the initial treatment with platinum (cPt)-based compounds, relapse is very common. We investigated the role of epigenetic changes in cPt-sensitive and -resistant EOC cell lines and found distinct differences in their enhancer landscape. Clinical data revealed that two genes (JAK1 and FGF10), which gained large enhancer clusters in resistant EOC cell lines, could provide novel biomarkers for early patient stratification with statistical independence for JAK1. To modulate the enhancer remodeling process and prevent the acquisition of cPt resistance in EOC cells, we performed a chromatin-focused RNAi screen in the presence of cPt. We identified subunits of the Nucleosome Remodeling and Deacetylase (NuRD) complex as critical factors sensitizing the EOC cell line A2780 to platinum treatment. Suppression of the Methyl-CpG Binding Domain Protein 3 (MBD3) sensitized cells and prevented the establishment of resistance under prolonged cPt exposure through alterations of H3K27ac at enhancer regions, which are differentially regulated in cPt-resistant cells, leading to a less aggressive phenotype. Our work establishes JAK1 as an independent prognostic marker and the NuRD complex as a potential target for combinational therapy.

Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics.

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell-ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell-ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two-photon stereolithography is adopted to print up to mm-sized high-precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein-based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two-pass printing or post-print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7-300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D-lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single-cell and tissue dynamics in response to defined mechanical and bio-molecular cues and is ultimately scalable to full organs.

OMTX705, a Novel FAP-Targeting ADC Demonstrates Activity in Chemotherapy and Pembrolizumab-Resistant Solid Tumor Models.

PURPOSE: The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy. EXPERIMENTAL DESIGN: We generated OMTX705, a novel antibody-drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin. Here, we studied its antineoplastic activity in vitro and in preclinical mouse models alone and in combination with chemotherapy as well as immunotherapy in PD-1-resistant tumors. RESULTS: In Avatar models, OMTX705 showed a 100% tumor growth inhibition and prolonged tumor regressions as single agent and in combination with chemotherapy. Treatment rechallenge following treatment discontinuation induced additional tumor regression, suggesting lack of treatment resistance. In a mouse model with a humanized immune system resistant to PD-1 inhibition, OMTX705 increased tumor infiltration by CD8+ T cells, induced complete regressions, and delayed tumor recurrence. CONCLUSIONS: These data suggest that FAP targeting with OMTX705 represents a novel and potent strategy for cancer treatment, including tumors resistant to immunotherapy, and support its clinical development.

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.

2-Methoxyestradiol Affects Mitochondrial Biogenesis Pathway and Succinate Dehydrogenase Complex Flavoprotein Subunit A in Osteosarcoma Cancer Cells.

BACKGROUND/AIM: Dysregulation of mitochondrial pathways is implicated in several diseases, including cancer. Notably, mitochondrial respiration and mitochondrial biogenesis are favored in some invasive cancer cells, such as osteosarcoma. Hence, the aim of the current work was to investigate the effects of 2-methoxyestradiol (2-ME), a potent anticancer agent, on the mitochondrial biogenesis of osteosarcoma cells. MATERIALS AND METHODS: Highly metastatic osteosarcoma 143B cells were treated with 2-ME separately or in combination with L-lactate, or with the solvent (non-treated control cells). Protein levels of α-syntrophin and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) were determined by western blotting. Impact of 2-ME on mitochondrial mass, regulation of cytochrome c oxidase I (COXI) expression, and succinate dehydrogenase complex flavoprotein subunit A (SDHA) was determined by immunofluorescence analyses. Inhibition of sirtuin 3 (SIRT3) activity by 2-ME was investigated by fluorescence assay and also, using molecular docking and molecular dynamics simulations. RESULTS: L-lactate induced mitochondrial biogenesis pathway via up-regulation of COXI. 2-ME inhibited mitochondrial biogenesis via regulation of PGC-1α, COXI, and SIRT3 in a concentration-dependent manner as a consequence of nuclear recruitment of neuronal nitric oxide synthase and nitric oxide generation. It was also proved that 2-ME inhibited SIRT3 activity by binding to both the canonical and allosteric inhibitor binding sites. Moreover, regardless of the mitochondrial biogenesis pathway, 2-ME affected the expression of SDHA. CONCLUSION: Herein, mitochondrial biogenesis pathway regulation and SDHA were presented as novel targets of 2-ME, and moreover, 2-ME was demonstrated as a potent inhibitor of SIRT3. L-lactate was confirmed to exert pro-carcinogenic effects on osteosarcoma cells via the induction of the mitochondrial biogenesis pathway. Thus, L-lactate level may be considered as a prognostic biomarker for osteosarcoma.

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.

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

Before entering mitosis, the stacks of the Golgi cisternae are separated from each other, and inhibiting this process delays entry of mammalian cells into mitosis. Protein kinase D (PKD) is known to be involved in Golgi-to-cell surface transport by controlling the biogenesis of specific transport carriers. Here we show that depletion of PKD1 and PKD2 proteins from HeLa cells by small interfering RNA leads to the accumulation of cells in the G2 phase of the cell cycle and prevents cells from entering mitosis. We further provide evidence that inhibition of PKD blocks mitotic Raf-1 and mitogen-activated protein kinase kinase (MEK) activation, and, as a consequence, mitotic Golgi fragmentation, which could be rescued by expression of active MEK1. Finally, Golgi fluorescence recovery after photobleaching analyses demonstrate that PKD is crucial for the cleavage of the noncompact zones of Golgi membranes in G2 phase. Our findings suggest that PKD controls interstack Golgi connections in a Raf-1/MEK1-dependent manner, a process required for entry of the cells into mitosis.

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 reporter's 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. See accompanying commentary by Gautam DOI: 10.1002/biot.201100424.