Protein Kinase D Isoforms Differentially Modulate Cofilin-Driven Directed Cell Migration

Background

Protein kinase D (PKD) enzymes regulate cofilin-driven actin reorganization and directed cell migration through both p21-activated kinase 4 (PAK4) and the phosphatase slingshot 1L (SSH1L). The relative contributions of different endogenous PKD isoforms to both signaling pathways have not been elucidated, sufficiently.

Methodology/Principal Findings

We here analyzed two cell lines (HeLa and MDA-MB-468) that express the subtypes protein kinase D2 (PKD2) and protein kinase D3 (PKD3). We show that under normal growth conditions both isoforms can form a complex, in which PKD3 is basally-active and PKD2 is inactive. Basal activity of PKD3 mediates PAK4 activity and downstream signaling, but does not significantly inhibit SSH1L. This signaling constellation was required for facilitating directed cell migration. Activation of PKD2 and further increase of PKD3 activity leads to additional phosphorylation and inhibition of endogenous SSH1L. Net effect is a dramatic increase in phospho-cofilin and a decrease in cell migration, since now both PAK4 and SSH1L are regulated by the active PKD2/PKD3 complex.

Conclusions/Significance

Our data suggest that PKD complexes provide an interface for both cofilin regulatory pathways. Dependent on the activity of involved PKD enzymes signaling can be balanced to guarantee a functional cofilin activity cycle and increase cell migration, or imbalanced to decrease cell migration. Our data also provide an explanation of how PKD isoforms mediate different effects on directed cell migration.     

Ring-like distribution of constitutive heterochromatin in bovine senescent cells

Background

Cells that reach “Hayflick limit” of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining.

Methods

We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH).

Results

Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli.

Conclusions

Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.     

Nuclear-targeted deleted in liver cancer 1 (DLC1) is less efficient in exerting its tumor suppressive activity both in vitro and in vivo

Background

Deleted in liver cancer 1 (DLC1) serves as an important RhoGTPase activating protein (RhoGAP) protein that terminates active RhoA signaling in human cancers. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends not only on RhoGAP activity, but also relies on proper focal adhesion localization through its interaction with tensin family proteins. Recently, there are reports showing that DLC1 can also be found in the nucleus; however, the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed.

Methodology and Principal Findings

We herein provide new evidence that DLC1 protein, which predominantly associated with focal adhesions and localized in cytosol, dynamically shuttled between cytoplasm and nucleus. Treatment of cells with nuclear export blocker, Leptomycin B (LMB), retained DLC1 in the nucleus. To understand the nuclear entry of DLC1, we identified amino acids 600–700 of DLC1 as a novel region that is important for its nuclear localization. The tumor suppressive activity of nuclear DLC1 was directly assessed by employing a nuclear localization signal (NLS) fusion variant of DLC1 (NLS-DLC1) with preferential nuclear localization. In SMMC-7721 HCC cells, expression of NLS-DLC1 failed to suppress colony formation and actin stress fiber formation in vitro. The abrogated tumor suppressive activity of nuclear DLC1 was demonstrated for the first time in vivo by subcutaneously injecting p53^−/− RasV12 hepatoblasts with stable NLS-DLC1 expression in nude mice. The injected hepatoblasts with NLS-DLC1 expression effectively formed tumors when compared with the non-nuclear targeted DLC1.

Conclusions/Significance

Our study identified a novel region responsible for the nuclear entry of DLC1 and demonstrated the functional difference of DLC1 in different cellular compartments both in vitro and in vivo.     

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background

Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified.

Methodology/Principal Findings

Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci.

Conclusions/Significance

This work provides the first clear evidence for a role for SIMs in SUMO E3 activity.     

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

Background

Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues.

Results

In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis.

Conclusions

Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.     

Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs

Background

6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases.

Methodology/Principal Findings

Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome.

Conclusion/Significance

6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.     

The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1

Background

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.

Principal Findings

As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.

Conclusions

ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.     

ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

Objective

The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1.

Research Design and Methods

L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity.

Results

In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with G_α-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL.

Conclusions

These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction.     

Altered expression of protein tyrosine phosphatase,non-receptor type 22 isoforms in systemic lupus erythematosus

Introduction

A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human protein tyrosine phosphatase, non-receptor type 22 (PTPN22) complementary DNA (cDNA) is associated with an increased risk of systemic lupus erythematosus (SLE). How the overall activity of PTPN22 is regulated and how the expression of PTPN22 differs between healthy individuals and patients with lupus are poorly understood. Our objectives were to identify novel alternatively spliced forms of PTPN22 and to examine the expression of PTPN22 isoforms in healthy donors and patients with lupus.

Methods

Various human PTPN22 isoforms were identified from the GenBank database or amplified directly from human T cells. The expression of these isoforms in primary T cells and macrophages was examined with real-time polymerase chain reaction. The function of the isoforms was determined with luciferase assays. Blood samples were collected from 49 subjects with SLE and 15 healthy controls. Correlation between the level of PTPN22 isoforms in peripheral blood and clinical features of SLE was examined with statistical analyses.

Results

Human PTPN22 was expressed in several isoforms, which differed in their level of expression and subcellular localization. All isoforms except one were functionally interchangeable in regulating NFAT activity. SLE patients expressed higher levels of PTPN22 than healthy individuals and the levels of PTPN22 were negatively correlated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC-DI).

Conclusions

The overall activity of PTPN22 is determined by the functional balance among all isoforms. The levels of PTPN22 isoforms in peripheral blood could represent a useful biomarker of SLE.     

Nutritional status modulates box C/D snoRNP biogenesis by regulated subcellular relocalization of the R2TP complex

Background

Box C/D snoRNPs, which are typically composed of box C/D snoRNA and the four core protein components Nop1, Nop56, Nop58, and Snu13, play an essential role in the modification and processing of pre-ribosomal RNA. The highly conserved R2TP complex, comprising the proteins Rvb1, Rvb2, Tah1, and Pih1, has been shown to be required for box C/D snoRNP biogenesis and assembly; however, the molecular basis of R2TP chaperone-like activity is not yet known.

Results

Here, we describe an unexpected finding in which the activity of the R2TP complex is required for Nop58 protein stability and is controlled by the dynamic subcellular redistribution of the complex in response to growth conditions and nutrient availability. In growing cells, the complex localizes to the nucleus and interacts with box C/D snoRNPs. This interaction is significantly reduced in poorly growing cells as R2TP predominantly relocalizes to the cytoplasm. The R2TP-snoRNP interaction is mainly mediated by Pih1.

Conclusions

The R2TP complex exerts a novel regulation on box C/D snoRNP biogenesis that affects their assembly and consequently pre-rRNA maturation in response to different growth conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0404-4) contains supplementary material, which is available to authorized users.     

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

Background

During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development.

Methodology/Principal Findings

Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition.

Conclusions/Significance

Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.     

Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background

The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues.

Methods

We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay.

Results

The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR.

Conclusions

RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.     

Tamoxifen Enhances the Hsp90 Molecular Chaperone ATPase Activity

Background

Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.

Methodology/Principal Findings

Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.

Conclusions/Significance

Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.     

Chronic Restraint Stress Inhibits Hair Growth via Substance P Mediated by Reactive Oxygen Species in Mice

Backgrounds

Solid evidence has demonstrated that psychoemotional stress induced alteration of hair cycle through neuropeptide substance P (SP) mediated immune response, the role of reactive oxygen species (ROS) in brain-skin-axis regulation system remains unknown.

Objectives

The present study aims to investigate possible mechanisms of ROS in regulation of SP-mast cell signal pathway in chronic restraint stress (CRS, a model of chronic psychoemotional stress) which induced abnormal of hair cycle.

Methods and Results

Our results have demonstrated that CRS actually altered hair cycle by inhibiting hair follicle growth in vivo, prolonging the telogen stage and delaying subsequent anagen and catagen stage. Up-regulation of SP protein expression in cutaneous peripheral nerve fibers and activation of mast cell were observed accompanied with increase of lipid peroxidation levels and reduction of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in CRS mice skin. In addition, SP receptor antagonist (RP67580) reduced mast cell activations and lipid peroxidation levels as well as increased GSH-Px activity and normalized hair cycle. Furthermore, antioxidant Tempol (a free radical scavenger) also restored hair cycle, reduced SP protein expression and mast cell activation.

Conclusions

Our study provides the first solid evidence for how ROS play a role in regulation of psychoemotional stress induced SP-Mast cell pathway which may provide a convincing rationale for antioxidant application in clinical treatment with psychological stress induced hair loss.     

Anti-Retroviral Therapy Decreases but Does Not Normalize Indoleamine 2,3-Dioxygenase Activity in HIV-Infected Patients

Background

Indoleamine 2,3-dioxygenase (IDO), which is mainly expressed in activated dendritic cells, catabolizes tryptophan to kynurenine and other downstream catabolites. It is known to be an immune mediator in HIV pathogenesis. The impact of anti-retroviral therapy on its activity has not been well established.

Methods

We measured systemic IDO activity (the ratio of plasma kynurenine to tryptophan) in HIV-infected patients before and after highly active antiretroviral therapy (HAART) and its association with a microbial translocation marker, soluble CD14 (sCD14).

Results

Among 76 participants, higher baseline IDO activity was associated with lower CD4⁺ T cell counts (P<0.05) and higher plasma sCD14 levels (P<0.001). After 1 year of HAART, IDO activity decreased significantly (P<0.01), but was still higher than in healthy controls (P<0.05). The baseline IDO activity did not predict CD4⁺ T cell recovery after 1 year of therapy. The percentages of myeloid and plasmacytoid dendritic cells were not correlated with IDO activity.

Conclusions

IDO activity is elevated in HIV-infected patients, which is partially associated with microbial translocation. HAART reduced, but did not normalize the activity of IDO.