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Thierry Dubois

Publications and source records attributed to Thierry Dubois.

14 recordsLinked to original sources
PubMed2006

Host protein interactions with enteropathogenic Escherichia coli (EPEC): 14-3-3tau binds Tir and has a role in EPEC-induced actin polymerization.

Enteropathogenic Escherichia coli (EPEC) cause infantile diarrhoea and are characterized by their ability to produce attaching and effacing lesions on the surface of intestinal epithelial cells. EPEC employ a filamentous type III secretion system to deliver effector molecules that subvert mammalian cell function to generate actin- and cytokeratin-rich pedestals beneath adherent bacteria. Tir is a major effector protein that is delivered to the plasma membrane of the eukaryotic cell where it acts as the receptor for the bacterial adhesin intimin. Host cell proteins that are recruited to the site of intimate attachment include focal adhesion and cytoskeletal proteins that contribute to pedestal formation. We have used Tir as bait in a yeast two-hybrid screen to identify the protein 14-3-3tau as a binding partner. 14-3-3 proteins are a family of adaptor proteins that modulate protein function in all eukaryotic cells. Here we demonstrate that the tau isoform (also known as theta) of 14-3-3 can bind specifically to Tir in a phosphorylation-independent manner, and that the interaction occurs during the infection process by co-immunoprecipitation of the partners from infected HeLa cell extracts. 14-3-3tau is recruited to the site of the pedestal (3 h after infection) and can decorate attached EPEC in the later stages of the infection process (5-7 h). Pedestal formation can be impaired by depletion of cellular 14-3-3tau using small interfering RNAs. This study indicates a direct functional role for the 14-3-3tau:Tir interaction and is the first to demonstrate the association of a host protein with the surface of EPEC.

14-3-3 Proteins↗
PubMed2005

Optical techniques for real-time penetration monitoring for laser welding.

Optical techniques for real-time full-penetration monitoring for Nd:YAG laser welding have been investigated. Coaxial light emission from the keyhole is imaged onto three photodiodes and a camera. We describe the spectral and statistical analyses from photodiode signals, which indicate the presence of a full penetration. Two image processing techniques based on the keyhole shape recognition and the keyhole image intensity profile along the welding path are presented. An intensity ratio parameter is used to determine the extent of opening at the rear of a fully opened keyhole. We show that this parameter clearly interprets a hole in formation or a lack of penetration when welding is performed on workpieces with variable thicknesses at constant laser power.

Journal Article↗
PubMed2005

Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics.

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.

ADP-Ribosylation Factor 1↗
PubMed2005

Closed-loop power and focus control of laser welding for full-penetration monitoring.

We describe a closed-loop control system ensuring full penetration in welding by controlling the focus position and power of a 4-kW Nd:YAG laser. A focus position monitoring system was developed based on the chromatic aberration of the focusing optics. With the laser power control system we can determine the degree of penetration by analyzing the keyhole image intensity profile. We demonstrate performance in bead-on-plate welding of Inconel 718 and titanium. The focus control system maintained a focal position on tilted and nonflat workpieces, and the penetration monitoring technique successfully controlled the laser power to maintain the full-penetration regime in the presence of linear and step changes of thickness. Finally we discuss the performances and the limits of the systems when applied to a realistic complex aerospace component.

Journal Article↗
PubMed2004

Association of CPI-17 with protein kinase C and casein kinase I.

The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.

Animals↗
PubMed2003

ARF6 controls post-endocytic recycling through its downstream exocyst complex effector.

The small guanosine triphosphate (GTP)-binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP-bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

ADP-Ribosylation Factor 6↗
PubMed2003

Centaurin-alpha(1) associates with and is phosphorylated by isoforms of protein kinase C.

Centaurin-alpha(1) is a member of the family of ADP-ribosylation factors (ARF) GTPase activating proteins (GAPs), although ARF GAP activity has not yet been demonstrated. The human homologue, centaurin-alpha(1) functionally complements the ARF GAP activity of Gcs1 in yeast. Although Gcs1 is involved in the formation of actin filaments in vivo, the function of centaurin remains elusive. We have identified a number of novel centaurin-alpha(1) binding partners; including CKIalpha and nucleolin. In this report, we have focused on the interaction of centaurin-alpha(1) with PKC. All groups of PKC associate directly through their cysteine rich domains. Centaurin-alpha(1) is also a substrate for all PKC classes and we have identified the two sites of phosphorylation. This is the first report of a kinase that phosphorylates centaurin-alpha(1).

Adaptor Proteins, Signal Transducing↗
PubMed2003

ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages.

Engulfment of particles by phagocytes is induced by their interaction with specific receptors on the cell surface, which leads to actin polymerization and the extension of membrane protrusions to form a closed phagosome. Membrane delivery from internal pools is considered to play an important role in pseudopod extension during phagocytosis. Here, we report that endogenous ADP ribosylation factor 6 (ARF6), a small GTP-binding protein, undergoes a sharp and transient activation in macrophages when phagocytosis was initiated via receptors for the Fc portion of immunoglobulins (FcRs). A dominant-negative mutant of ARF6 (T27N mutation) dramatically affected FcR-mediated phagocytosis. Expression of ARF6-T27N lead to a reduction in the focal delivery of vesicle-associated membrane protein 3+ endosomal recycling membranes at phagocytosis sites, whereas actin polymerization was unimpaired. This resulted in an early blockade in pseudopod extension and accumulation of intracellular vesicles, as observed by electron microscopy. We conclude that ARF6 is a major regulator of membrane recycling during phagocytosis.

ADP-Ribosylation Factor 6↗
PubMed2003

Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17.

CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.

Brain↗
PubMed2003

Centaurin-alpha 1 associates in vitro and in vivo with nucleolin.

Centaurin-alpha(1) was originally described as a binding partner for phosphoinositides. In spite of the presence of a putative ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) domain, no ARF-GAP activity has been attributed to centaurin-alpha(1) so far. Thus the function of this protein remains to be determined. In order to better understand its intracellular role, we aimed to identify centaurin-alpha(1) partners. Using affinity chromatography followed by mass spectrometry analysis, we identified several potential centaurin-alpha(1) protein partners. Nucleolin, a nucleolar protein involved in ribosome biosynthesis, was the main centaurin-alpha(1) interacting protein. The interaction between centaurin-alpha(1) and nucleolin was confirmed by Western blot analysis and GST pull down assays. Moreover, we have shown that ectopically expressed centaurin-alpha(1) associates in vivo with endogenous nucleolin in human embryonic kidney 293 cells. In addition, the association between nucleolin and centaurin-alpha(1) was disrupted by RNAse treatment, indicating that RNA integrity was necessary for their binding. This suggested that centaurin-alpha(1) was part of a ribonucleoprotein complex.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells.

PCTAIRE-1 is a CDK-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Bacterially expressed PCTAIRE is completely inactive as a protein kinase, but is a very good substrate for protein kinase A (PKA), which phosphorylates a total of four sites in the N-terminus of PCTAIRE-1. Phosphorylation of one of these sites, Ser119, generates a 14-3-3 binding site, which is functional in vitro as well as in vivo. Mutation of another PKA site, Ser153, to an alanine residue generated an activated kinase in transfected mammalian cells. This activity was comparable to that of CDK5 activated by a bacterially expressed, truncated version of p35(nck), p21. Gel filtration analysis of a brain extract suggested that monomeric PCTAIRE-1 was the active species, implying that PCTAIRE-1 may not be a true CDK, in that it does not require a partner (cyclin-like) subunit for kinase activity. Finally, we found that various forms of PCTAIRE-1 transfected into neuroblastoma cell lines could either promote or inhibit neurite outgrowth, suggesting a potential role for the PCTAIRE-1 gene product in the control of neurite outgrowth.

Amino Acid Sequence↗
PubMed2002

Identification of casein kinase Ialpha interacting protein partners.

Casein kinase Ialpha (CKIalpha) belongs to a family of serine/threonine protein kinases involved in membrane trafficking, RNA processing, mitotic spindle formation and cell cycle progression. In this report, we identified several CKIalpha interacting proteins including RCC1, high mobility group proteins 1 and 2 (HMG1, HMG2), Erf, centaurin-alpha1, synaptotagmin IX and CPI-17 that were isolated from brain as CKIalpha co-purifying proteins. Actin, importin-alpha(1), importin-beta, PP2Ac, centaurin-alpha1, and HMG1 were identified by affinity chromatography using a peptide column comprising residues 214-233 of CKIalpha. We have previously shown that centaurin-alpha1 represents a CKIalpha partner both in vitro and in vivo. The nuclear protein regulator of chromosome condensation 1 (RCC1) is a guanosine nucleotide exchange factor for Ran which is involved in nuclear transport and mitotic spindle formation. Here we show that CKIalpha and RCC1 interact in brain and in cultured cells. However, the interaction does not involve residues 217-233 of CKIalpha which are proposed from X-ray structures to represent an anchoring site for CKI partners. Formation of the RCC1/CKIalpha complex is consistent with the association of the kinase with mitotic spindles. In conclusion, we have identified a number of novel CKIalpha protein partners and their relations to CKI are discussed.

Actins↗
PubMed2002

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.

Amino Acid Sequence↗