Pierre Lutz - Academia.edu (original) (raw)

Papers by Pierre Lutz

Research paper thumbnail of Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter

Journal of virology, 1996

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication... more The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the b...

Research paper thumbnail of Interaction of the adenovirus L1 52/55-kilodalton protein with the IVa2 gene product during infection

Journal of virology, 1996

The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, ... more The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, raising the possibility that it has multiple roles in the viral life cycle. To obtain possible insights into these roles, the yeast two-hybrid system was used to examine the interactions of the 52/55-kDa protein with viral and cellular factors. cDNA expression libraries from human 293 cells at both early and late stages of adenovirus type 5 infection were constructed and screened, with the 52/55-kDa protein being used as bait. Characterization of positive clones revealed that the adenovirus IVa2 gene product interacted specifically with the 52/55-kDa protein. In addition, the IVa2 protein was shown to interact with a bacterial glutathione S-transferase-52/55-kDa fusion protein in vitro, further supporting the finding with the yeast two-hybrid system. Finally, coimmunoprecipitation studies confirmed that the 52/55-kDa protein and IVa2 polypeptide interact specifically during the course of...

Research paper thumbnail of Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter

Journal of virology, 1996

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication... more The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the b...

Research paper thumbnail of Interaction of the adenovirus L1 52/55-kilodalton protein with the IVa2 gene product during infection

Journal of virology, 1996

The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, ... more The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, raising the possibility that it has multiple roles in the viral life cycle. To obtain possible insights into these roles, the yeast two-hybrid system was used to examine the interactions of the 52/55-kDa protein with viral and cellular factors. cDNA expression libraries from human 293 cells at both early and late stages of adenovirus type 5 infection were constructed and screened, with the 52/55-kDa protein being used as bait. Characterization of positive clones revealed that the adenovirus IVa2 gene product interacted specifically with the 52/55-kDa protein. In addition, the IVa2 protein was shown to interact with a bacterial glutathione S-transferase-52/55-kDa fusion protein in vitro, further supporting the finding with the yeast two-hybrid system. Finally, coimmunoprecipitation studies confirmed that the 52/55-kDa protein and IVa2 polypeptide interact specifically during the course of...

Research paper thumbnail of The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer

Cancer Immunology Research, 2019

The escape of cancer cells from host immunosurveillance involves a shift in immune responses, inc... more The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in T helper 1 (Th1) and Th2 cells. A Th1-dominated immune response predicts positive outcome in colorectal cancer (CRC). The E3 ubiquitin ligase Asb2α is expressed in Th2 cells, but its roles in T cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator Gata3 induces Asb2. Loss of Asb2 did not affect Th differentiation ex vivo, but reduced Il-4 production from Th2 cells. We found that high ASB2 levels associated with poor outcome in CRC. Importantly, loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitisassociated tumorigenesis in mice. Diminished Th2 function correlated with increased IFN-γ production and an enhanced type 1 anti-tumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.

Research paper thumbnail of The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer

Cancer Immunology Research, 2019

The escape of cancer cells from host immunosurveillance involves a shift in immune responses, inc... more The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in T helper 1 (Th1) and Th2 cells. A Th1-dominated immune response predicts positive outcome in colorectal cancer (CRC). The E3 ubiquitin ligase Asb2α is expressed in Th2 cells, but its roles in T cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator Gata3 induces Asb2. Loss of Asb2 did not affect Th differentiation ex vivo, but reduced Il-4 production from Th2 cells. We found that high ASB2 levels associated with poor outcome in CRC. Importantly, loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitisassociated tumorigenesis in mice. Diminished Th2 function correlated with increased IFN-γ production and an enhanced type 1 anti-tumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.

Research paper thumbnail of leukemia cells growth-arrested by retinoic acid Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid

Research paper thumbnail of Filamins Regulate Cell Spreading and Initiation of Cell Migration

Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding ... more Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding member of the family, was implicated in migration by cell biological analyses and the identification of FLNA mutations in the neuronal migration disorder periventricular heterotopia. However, recent knockout studies have questioned the relevance of FLNa to cell migration. Here we have used shRNA-mediated knockdown of FLNa, FLNb or FLNa and FLNb, or, alternatively, acute proteasomal degradation of all three FLNs, to generate FLN-deficient cells and assess their ability to migrate. We report that loss of FLNa or FLNb has little effect on migration but that knockdown of FLNa and FLNb, or proteolysis of all three FLNs, impairs migration. The observed defect is primarily a deficiency in initiation of motility rather than a problem with maintenance of locomotion speed. FLN-deficient cells are also impaired in spreading. Re-expression of full length FLNa, but not re-expression of a mutated FLNa lacking immunoglobulin domains 19 to 21, reverts both the spreading and the inhibition of initiation of migration. Our results establish a role for FLNs in cell migration and spreading and suggest that compensation by other FLNs may mask phenotypes in single knockout or knockdown cells. We propose that interactions between FLNs and transmembrane or signalling proteins, mediated at least in part by immunoglobulin domains 19 to 21 are important for both cell spreading and initiation of migration.

Research paper thumbnail of Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

PLoS ONE, 2012

The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hemato... more The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates.

Research paper thumbnail of Functional and Structural Insights into ASB2 , a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells

Journal of Biological Chemistry, 2011

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins... more By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor ␣ oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2␣) and a muscle-type (ASB2␤) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2␣ is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2␣ structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2␣, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ␤1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2␣, together with ankyrin repeats 1 to 10, is necessary for association of ASB2␣ with filamin A. Importantly, the ASB2␣ N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ␤ integrins. Together, these data provide new insights into the molecular mechanisms of ASB2␣ binding to filamin.

Research paper thumbnail of Ubiquitin-mediated proteasomal degradation in normal and malignant hematopoiesis

Blood Cells, Molecules, and Diseases, 2008

Understanding the molecular mechanisms controlling normal hematopoietic differentiation is critic... more Understanding the molecular mechanisms controlling normal hematopoietic differentiation is critical to develop new treatments for blood diseases and to manipulate stem cells. Despite the identification of many players in hematopoiesis, the molecular mechanisms controlling hematopoietic differentiation remain poorly understood. Due to a number of recent findings, the targeting of regulators of hematopoiesis to proteasomal degradation might be an important step in control of this developmental program.

Research paper thumbnail of ASB2  regulates migration of immature dendritic cells

Blood, 2013

Key Points By demonstrating a novel mechanism of regulation of FLN stability by ASB2α, our result... more Key Points By demonstrating a novel mechanism of regulation of FLN stability by ASB2α, our results point to FLNs and ASB2α as new players in DC biology. Our data highlight a new degree of complexity in the events that regulate cell motility of immature DCs.

Research paper thumbnail of Molecular Tuning of Filamin A Activities in the Context of Adhesion and Migration

Frontiers in Cell and Developmental Biology, 2020

The dynamic organization of actin cytoskeleton meshworks relies on multiple actin-binding protein... more The dynamic organization of actin cytoskeleton meshworks relies on multiple actin-binding proteins endowed with distinct actin-remodeling activities. Filamin A is a large multi-domain scaffolding protein that cross-links actin filaments with orthogonal orientation in response to various stimuli. As such it plays key roles in the modulation of cell shape, cell motility, and differentiation throughout development and adult life. The essentiality and complexity of Filamin A is highlighted by mutations that lead to a variety of severe human disorders affecting multiple organs. One of the most conserved activity of Filamin A is to bridge the actin cytoskeleton to integrins, thereby maintaining the later in an inactive state. We here review the numerous mechanisms cells have developed to adjust Filamin A content and activity and focus on the function of Filamin A as a gatekeeper to integrin activation and associated adhesion and motility.

Research paper thumbnail of Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

PLoS ONE, 2012

The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hemato... more The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. Citation: Lamsoul I, Erard M, van der Ven PFM, Lutz PG (2012) Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells. PLoS ONE 7(8): e43798.

Research paper thumbnail of Functional and Structural Insights into ASB2 , a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells

Journal of Biological Chemistry, 2011

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins... more By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor ␣ oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2␣) and a muscle-type (ASB2␤) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2␣ is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2␣ structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2␣, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ␤1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2␣, together with ankyrin repeats 1 to 10, is necessary for association of ASB2␣ with filamin A. Importantly, the ASB2␣ N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ␤ integrins. Together, these data provide new insights into the molecular mechanisms of ASB2␣ binding to filamin.

Research paper thumbnail of Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid

A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a ... more A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mecha- nisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblas- tin is involved in the

Research paper thumbnail of Substrates of the ASB2α E3 ubiquitin ligase in dendritic cells

Scientific Reports, 2015

Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functio... more Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.

Research paper thumbnail of Nucleoplasmic and Nucleolar Distribution of the Adenovirus IVa2 Gene Product

Sequence elements (DE) located downstream of the adenovirus major late promoter start site have p... more Sequence elements (DE) located downstream of the adenovirus major late promoter start site have previ- ously been shown to be essential for the activation of this promoter after the onset of viral DNA replication. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner and contribute to thisactivation.DEF-BcorrespondstoadimeroftheadenovirusIVa2geneproduct(pIVa2,449residues),while DEF-A is a heteromeric protein also comprising pIVa2. As

Research paper thumbnail of JAML, a novel protein with characteristics of a junctional adhesion molecule, is induced during differentiation of myeloid leukemia cells

Retinoic acid induces clinical remission in acute promyelocytic leukemia (APL) by triggering diff... more Retinoic acid induces clinical remission in acute promyelocytic leukemia (APL) by triggering differentiation of leukemia pro- myelocytes. Here, we have characterized a gene encoding a member of the immuno- globulin superfamily, among novel reti- noic acid-induced genes identified in APL cells. This protein, which was named JAML (junctional adhesion molecule- like), contains 2 extracellular immuno- globulin-like domains, a transmembrane segment,

Research paper thumbnail of Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells

Proceedings of the National Academy of Sciences, 2000

Hematopoiesis depends on a pool of quiescent hematopoietic stem͞progenitor cells. When exposed to... more Hematopoiesis depends on a pool of quiescent hematopoietic stem͞progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colonystimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factorindependent growth to murine bone marrow-derived Ba͞F3͞G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C͞EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C͞EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.

Research paper thumbnail of Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter

Journal of virology, 1996

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication... more The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the b...

Research paper thumbnail of Interaction of the adenovirus L1 52/55-kilodalton protein with the IVa2 gene product during infection

Journal of virology, 1996

The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, ... more The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, raising the possibility that it has multiple roles in the viral life cycle. To obtain possible insights into these roles, the yeast two-hybrid system was used to examine the interactions of the 52/55-kDa protein with viral and cellular factors. cDNA expression libraries from human 293 cells at both early and late stages of adenovirus type 5 infection were constructed and screened, with the 52/55-kDa protein being used as bait. Characterization of positive clones revealed that the adenovirus IVa2 gene product interacted specifically with the 52/55-kDa protein. In addition, the IVa2 protein was shown to interact with a bacterial glutathione S-transferase-52/55-kDa fusion protein in vitro, further supporting the finding with the yeast two-hybrid system. Finally, coimmunoprecipitation studies confirmed that the 52/55-kDa protein and IVa2 polypeptide interact specifically during the course of...

Research paper thumbnail of Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter

Journal of virology, 1996

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication... more The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the b...

Research paper thumbnail of Interaction of the adenovirus L1 52/55-kilodalton protein with the IVa2 gene product during infection

Journal of virology, 1996

The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, ... more The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, raising the possibility that it has multiple roles in the viral life cycle. To obtain possible insights into these roles, the yeast two-hybrid system was used to examine the interactions of the 52/55-kDa protein with viral and cellular factors. cDNA expression libraries from human 293 cells at both early and late stages of adenovirus type 5 infection were constructed and screened, with the 52/55-kDa protein being used as bait. Characterization of positive clones revealed that the adenovirus IVa2 gene product interacted specifically with the 52/55-kDa protein. In addition, the IVa2 protein was shown to interact with a bacterial glutathione S-transferase-52/55-kDa fusion protein in vitro, further supporting the finding with the yeast two-hybrid system. Finally, coimmunoprecipitation studies confirmed that the 52/55-kDa protein and IVa2 polypeptide interact specifically during the course of...

Research paper thumbnail of The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer

Cancer Immunology Research, 2019

The escape of cancer cells from host immunosurveillance involves a shift in immune responses, inc... more The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in T helper 1 (Th1) and Th2 cells. A Th1-dominated immune response predicts positive outcome in colorectal cancer (CRC). The E3 ubiquitin ligase Asb2α is expressed in Th2 cells, but its roles in T cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator Gata3 induces Asb2. Loss of Asb2 did not affect Th differentiation ex vivo, but reduced Il-4 production from Th2 cells. We found that high ASB2 levels associated with poor outcome in CRC. Importantly, loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitisassociated tumorigenesis in mice. Diminished Th2 function correlated with increased IFN-γ production and an enhanced type 1 anti-tumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.

Research paper thumbnail of The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer

Cancer Immunology Research, 2019

The escape of cancer cells from host immunosurveillance involves a shift in immune responses, inc... more The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in T helper 1 (Th1) and Th2 cells. A Th1-dominated immune response predicts positive outcome in colorectal cancer (CRC). The E3 ubiquitin ligase Asb2α is expressed in Th2 cells, but its roles in T cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator Gata3 induces Asb2. Loss of Asb2 did not affect Th differentiation ex vivo, but reduced Il-4 production from Th2 cells. We found that high ASB2 levels associated with poor outcome in CRC. Importantly, loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitisassociated tumorigenesis in mice. Diminished Th2 function correlated with increased IFN-γ production and an enhanced type 1 anti-tumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.

Research paper thumbnail of leukemia cells growth-arrested by retinoic acid Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid

Research paper thumbnail of Filamins Regulate Cell Spreading and Initiation of Cell Migration

Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding ... more Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding member of the family, was implicated in migration by cell biological analyses and the identification of FLNA mutations in the neuronal migration disorder periventricular heterotopia. However, recent knockout studies have questioned the relevance of FLNa to cell migration. Here we have used shRNA-mediated knockdown of FLNa, FLNb or FLNa and FLNb, or, alternatively, acute proteasomal degradation of all three FLNs, to generate FLN-deficient cells and assess their ability to migrate. We report that loss of FLNa or FLNb has little effect on migration but that knockdown of FLNa and FLNb, or proteolysis of all three FLNs, impairs migration. The observed defect is primarily a deficiency in initiation of motility rather than a problem with maintenance of locomotion speed. FLN-deficient cells are also impaired in spreading. Re-expression of full length FLNa, but not re-expression of a mutated FLNa lacking immunoglobulin domains 19 to 21, reverts both the spreading and the inhibition of initiation of migration. Our results establish a role for FLNs in cell migration and spreading and suggest that compensation by other FLNs may mask phenotypes in single knockout or knockdown cells. We propose that interactions between FLNs and transmembrane or signalling proteins, mediated at least in part by immunoglobulin domains 19 to 21 are important for both cell spreading and initiation of migration.

Research paper thumbnail of Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

PLoS ONE, 2012

The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hemato... more The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates.

Research paper thumbnail of Functional and Structural Insights into ASB2 , a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells

Journal of Biological Chemistry, 2011

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins... more By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor ␣ oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2␣) and a muscle-type (ASB2␤) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2␣ is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2␣ structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2␣, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ␤1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2␣, together with ankyrin repeats 1 to 10, is necessary for association of ASB2␣ with filamin A. Importantly, the ASB2␣ N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ␤ integrins. Together, these data provide new insights into the molecular mechanisms of ASB2␣ binding to filamin.

Research paper thumbnail of Ubiquitin-mediated proteasomal degradation in normal and malignant hematopoiesis

Blood Cells, Molecules, and Diseases, 2008

Understanding the molecular mechanisms controlling normal hematopoietic differentiation is critic... more Understanding the molecular mechanisms controlling normal hematopoietic differentiation is critical to develop new treatments for blood diseases and to manipulate stem cells. Despite the identification of many players in hematopoiesis, the molecular mechanisms controlling hematopoietic differentiation remain poorly understood. Due to a number of recent findings, the targeting of regulators of hematopoiesis to proteasomal degradation might be an important step in control of this developmental program.

Research paper thumbnail of ASB2  regulates migration of immature dendritic cells

Blood, 2013

Key Points By demonstrating a novel mechanism of regulation of FLN stability by ASB2α, our result... more Key Points By demonstrating a novel mechanism of regulation of FLN stability by ASB2α, our results point to FLNs and ASB2α as new players in DC biology. Our data highlight a new degree of complexity in the events that regulate cell motility of immature DCs.

Research paper thumbnail of Molecular Tuning of Filamin A Activities in the Context of Adhesion and Migration

Frontiers in Cell and Developmental Biology, 2020

The dynamic organization of actin cytoskeleton meshworks relies on multiple actin-binding protein... more The dynamic organization of actin cytoskeleton meshworks relies on multiple actin-binding proteins endowed with distinct actin-remodeling activities. Filamin A is a large multi-domain scaffolding protein that cross-links actin filaments with orthogonal orientation in response to various stimuli. As such it plays key roles in the modulation of cell shape, cell motility, and differentiation throughout development and adult life. The essentiality and complexity of Filamin A is highlighted by mutations that lead to a variety of severe human disorders affecting multiple organs. One of the most conserved activity of Filamin A is to bridge the actin cytoskeleton to integrins, thereby maintaining the later in an inactive state. We here review the numerous mechanisms cells have developed to adjust Filamin A content and activity and focus on the function of Filamin A as a gatekeeper to integrin activation and associated adhesion and motility.

Research paper thumbnail of Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

PLoS ONE, 2012

The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hemato... more The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. Citation: Lamsoul I, Erard M, van der Ven PFM, Lutz PG (2012) Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells. PLoS ONE 7(8): e43798.

Research paper thumbnail of Functional and Structural Insights into ASB2 , a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells

Journal of Biological Chemistry, 2011

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins... more By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor ␣ oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2␣) and a muscle-type (ASB2␤) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2␣ is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2␣ structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2␣, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ␤1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2␣, together with ankyrin repeats 1 to 10, is necessary for association of ASB2␣ with filamin A. Importantly, the ASB2␣ N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ␤ integrins. Together, these data provide new insights into the molecular mechanisms of ASB2␣ binding to filamin.

Research paper thumbnail of Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid

A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a ... more A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mecha- nisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblas- tin is involved in the

Research paper thumbnail of Substrates of the ASB2α E3 ubiquitin ligase in dendritic cells

Scientific Reports, 2015

Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functio... more Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.

Research paper thumbnail of Nucleoplasmic and Nucleolar Distribution of the Adenovirus IVa2 Gene Product

Sequence elements (DE) located downstream of the adenovirus major late promoter start site have p... more Sequence elements (DE) located downstream of the adenovirus major late promoter start site have previ- ously been shown to be essential for the activation of this promoter after the onset of viral DNA replication. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner and contribute to thisactivation.DEF-BcorrespondstoadimeroftheadenovirusIVa2geneproduct(pIVa2,449residues),while DEF-A is a heteromeric protein also comprising pIVa2. As

Research paper thumbnail of JAML, a novel protein with characteristics of a junctional adhesion molecule, is induced during differentiation of myeloid leukemia cells

Retinoic acid induces clinical remission in acute promyelocytic leukemia (APL) by triggering diff... more Retinoic acid induces clinical remission in acute promyelocytic leukemia (APL) by triggering differentiation of leukemia pro- myelocytes. Here, we have characterized a gene encoding a member of the immuno- globulin superfamily, among novel reti- noic acid-induced genes identified in APL cells. This protein, which was named JAML (junctional adhesion molecule- like), contains 2 extracellular immuno- globulin-like domains, a transmembrane segment,

Research paper thumbnail of Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells

Proceedings of the National Academy of Sciences, 2000

Hematopoiesis depends on a pool of quiescent hematopoietic stem͞progenitor cells. When exposed to... more Hematopoiesis depends on a pool of quiescent hematopoietic stem͞progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colonystimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factorindependent growth to murine bone marrow-derived Ba͞F3͞G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C͞EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C͞EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.