Talin regulates moesin–NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin–NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.     

Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9–Rad1–Hus1 complex

Rad9–Rad1–Hus1 (9–1–1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9–1–1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9–1–1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9–1–1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9–1–1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and β-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1–8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH₄. A trimeric human 9–1–1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9–1–1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9–1–1 reduced the formation of 8-oxoG residues following the H₂O₂ treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9–1–1. These indicate that individual human 9–1–1 subunits and human 9–1–1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H₂O₂-treated cells was derived from the 9–1–1 complex as a whole.     

Liprin-α2 promotes the presynaptic recruitment and turnover of RIM1/CASK to facilitate synaptic transmission

The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin–proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.     

Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1^−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1^−/− cells.     

Epstein-Barr virus BPLF1 deubiquitinates PCNA and attenuates polymerase η recruitment to DNA damage sites

PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity.     

Interaction between Duodenase and α1-Proteinase Inhibitor

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and ₁-proteinase inhibitor (₁-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and ₁-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·10⁵ M^–1·sec^–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and ₁-PI in identical compartments, ₁-PI is suggested to be a duodenase inhibitor in vivo.     

Thermodynamics of the Interaction between αs1- and κ-Caseins

Pyrenebutyrate-conjugated α_s1-casein was prepared and the complex formation between α_s1- and κ-casein polymers was investigated by fluorescence polarization. The complex formation was also investigated by a microcalorimetric technique. The positive enthalpy and entropy changes and endothermic nature suggested the hydrophobic interaction between α_s1- and κ-casein polymers.

The degree of polarization of κ-casein polymer decreased with the addition of 1-anilino-8-naphthalenesulfonate (ANS), while that of α_s1-casein polymer and α_s1-κ-casein complex was invariant. Moreover the reaction of κ-casein polymer and ANS was exothermic. These facts suggested that the intermolecular hydrophobic regions in κ-casein polymer were disrupted by the adsorption of ANS. The rotational relaxation time of pyrenebutyrate conjugated complex between cyanoethyl-κ-casein and α_s1-casein polymer was smaller than that of cyanoethyl-κ-casein alone. From these results, it was postulated that the dissociation of κ-casein polymer by the complex formation with α_s1-casein polymer might be caused by the disruption of the intermolecular hydrophobic bonds in κ-casein polymer.     

Interaction between Auxin–binding Protein–I and RNA Polymerase II

Interactions between auxin–binding protein–I (ABP–I), purified from etiolated mung bean seedlings, and nuclear components from mung bean tissues were investigated. When NaCI–solubilized components of chromatin were put on an affinity column of ABP–I–Iinked Sepharose 4B, a small amount of the material was retained on the affinity column and was eluted with 1 M NaCl. RNA polymerase II activity was detected in the eluted fraction. Partially purified RNA polymerase II from mung bean nuclei and purified RNA polymerase II from wheat germ also bound to ABP–I. Indole–3–acetic acid was not necessary for the binding of RNA polymerase II to ABP–I. Acid–denatured ABP–I did not bind to RNA polymerase II from wheat germ. The addition of ABP–I to the reaction mixture for RNA synthesis in vitro caused a stimulation of the activity of wheat germ RNA polymerase.     

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity

Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)-deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-β1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-β1 activity and decreased cell migration, an effect blocked by inhibiting TGF-β1. Furthermore, TGF-β-mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin-mediated activation of TGF-β. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-β activity and identify a novel function for endoglin in controlling pericyte behavior.     

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.     

Cystatin B and SOD1: Protein–Protein Interaction and Possible Relation to Neurodegeneration

Cystatin B (CSTB), an inhibitor of the cysteine proteases, belongs to the cathepsin family and it is known to interact with a number of proteins involved in cytoskeletal organization. CSTB has an intrinsic tendency to form aggregates depending on the redox environment. The gene encoding for CSTB is frequently mutated in association with the rare neurodegenerative condition progressive myoclonus epilepsy. Increased levels of CSTB have been observed in the spinal cord of transgenic mice modeling SOD1-linked familial amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motoneurons. In the present study, we have investigated the relationship occurring between the expression of SOD1 and CSTB either wild-type or double-cysteine substitution mutant (Cys 3 and Cys 64). Whether or not there is a physical interaction between the two proteins was also investigated in overexpression experiments using a human neuroblastoma cell line and mouse-immortalized motoneurons. Here we report evidences for a reciprocal influence of CSTB and SOD1 at the gene expression level and for a direct interaction of the two proteins.     

Mec1/Tel1-dependent phosphorylation of Slx4 stimulates Rad1–Rad10-dependent cleavage of non-homologous DNA tails

Budding yeast Slx4 interacts with the Rad1–Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single-strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3′ non-homologous (NH) DNA tails by Rad1–Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double-strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3′ NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified a specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1–Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR.     

FAM29A promotes microtubule amplification via recruitment of the NEDD1–γ-tubulin complex to the mitotic spindle

Microtubules (MTs) are nucleated from centrosomes and chromatin. In addition, MTs can be generated from preexiting MTs in a γ-tubulin–dependent manner in yeast, plant, and Drosophila cells, although the underlying mechanism remains unknown. Here we show the spindle-associated protein FAM29A promotes MT-dependent MT amplification and is required for efficient chromosome congression and segregation in mammalian cells. Depletion of FAM29A reduces spindle MT density. FAM29A is not involved in the nucleation of MTs from centrosomes and chromatin, but is required for a subsequent increase in MT mass in cells released from nocodazole. FAM29A interacts with the NEDD1–γ-tubulin complex and recruits this complex to the spindle, which, in turn, promotes MT polymerization. FAM29A preferentially associates with kinetochore MTs and knockdown of FAM29A reduces the number of MTs in a kinetochore fiber, activates the spindle checkpoint, and delays the mitotic progression. Our study provides a biochemical mechanism for MT-dependent MT amplification and for the maturation of kinetochore fibers in mammalian cells.     

Desmoglein 1–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca²⁺-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.     

Membrane transport of WAVE2 and lamellipodia formation require Pak1 that mediates phosphorylation and recruitment of stathmin/Op18 to Pak1–WAVE2–kinesin complex

Membrane transport of WAVE2 that leads to lamellipodia formation requires a small GTPase Rac1, the motor protein kinesin, and microtubules. Here we explore the possibility of whether the Rac1-dependent and kinesin-mediated WAVE2 transport along microtubules is regulated by a p21-activated kinase Pak as a downstream effector of Rac1. We find that Pak1 constitutively binds to WAVE2 and is transported with WAVE2 to the leading edge by stimulation with hepatocyte growth factor (HGF). Concomitantly, phosphorylation of tubulin-bound stathmin/Op18 at serine 25 (Ser25) and Ser38, microtubule growth, and stathmin/Op18 binding to kinesin–WAVE2 complex were induced. The HGF-induced WAVE2 transport, lamellipodia formation, stathmin/Op18 phosphorylation at Ser38 and binding to kinesin–WAVE2 complex, but not stathmin/Op18 phosphorylation at Ser25 and microtubule growth, were abrogated by Pak1 inhibitor IPA-3 and Pak1 depletion with small interfering RNA (siRNA). Moreover, stathmin/Op18 depletion with siRNA caused significant inhibition of HGF-induced WAVE2 transport and lamellipodia formation, with HGF-independent promotion of microtubule growth. Collectively, it is suggested that Pak1 plays a critical role in HGF-induced WAVE2 transport and lamellipodia formation by directing Pak1–WAVE2–kinesin complex toward the ends of growing microtubules through phosphorylation and recruitment of tubulin-bound stathmin/Op18 to the complex.