Valerie Lang - Academia.edu (original) (raw)

Papers by Valerie Lang

Molecular biology and evolution, Jan 4, 2015

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is ... more SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella...

The Journal of Immunology

4 Abbreviations used in this paper: SH2, Src homology 2 domain ; IS, immunological synapse; PtdIn... more 4 Abbreviations used in this paper: SH2, Src homology 2 domain ; IS, immunological synapse; PtdInsP 2, phosphatidylinositol-4,5-bisphosphate; PtdInsP 3, phosphatidylinositol-3,4,5trisphosphate; WT, wild type; WB, western blot.

Oncogene, 2015

Class IA phosphatidylinositol 3-kinases (PI3Ks) are composed of p110 catalytic and p85 regulatory... more Class IA phosphatidylinositol 3-kinases (PI3Ks) are composed of p110 catalytic and p85 regulatory subunits. How regulatory subunits modulate PI3K activity remains only partially understood. Here we identified SUMO (small ubiquitin-related modifier) as a new player modulating this regulation. We demonstrate that both p85β and p85α are conjugated to SUMO1 and SUMO2. We identified two lysine residues located at the inter-SH2 domain on p85β, a critical region required for inhibition of p110, as being required for SUMO conjugation. A SUMOylation-defective mutant p85β shows higher activation of the PI3K pathway, and increased cell migration and transformation. Moreover, the cancer-related KS459del mutant in p85α was less efficiently SUMOylated compared with the wild-type protein. Finally, our results show that SUMO modulates p85 tyrosine phosphorylation, a modification correlating with PI3K pathway activation. Thus, SUMO reduces the levels of tyrosine-phosphorylated-p85 while loss of SUMOylation results in increased tyrosine phosphorylation of p85. In summary, we identify SUMO as a new important player in the regulation of the PI3K pathway through modulation of p85.Oncogene advance online publication, 28 September 2015; doi:10.1038/onc.2015.356.

Molecular and Cellular Biology

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C te... more Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-kappaB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-kappaB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncog...

Cell death & disease, 2013

The zinc-finger protein A20 is a key player in the negative feedback regulation of the nuclear fa... more The zinc-finger protein A20 is a key player in the negative feedback regulation of the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) pathway in response to multiple stimuli. Tumor necrosis factor alpha (TNFα), a cytokine with pleiotropic effects on cellular proliferation and differentiation, dramatically increases A20 expression in all tissues. As TNFα inhibits adipocyte differentiation, we have determined the contribution of A20 to the adipogenic capacity of human mesenchymal stromal cells (MSCs). Here we show that A20 is constitutively expressed in MSCs, which previously has been observed only in cells that are either tumor or immune cells (T/B lymphocytes). TNFα stimulation induced a rapid degradation of A20 protein mediated exclusively by the proteasome in MSCs and not by caspases. This degradation is concomitant to the induction of its own mRNA, which suggests that a tight regulation of NF-κB signaling in MSCs is fundamental. On one hand, we demonstrate ...

Molecular and cellular biology, 2003

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of... more NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each ...

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1999

A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src prot... more A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized ...

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

CD5 is a 67-kDa T cell surface Ag that can be found physically associated with the CD3-TCR molecu... more CD5 is a 67-kDa T cell surface Ag that can be found physically associated with the CD3-TCR molecular complex. In different experimental models it has been shown to act as a costimulatory receptor for T cell activation. Unexpectedly, studies in CD5-deficient mice suggested a negative role for the CD5 Ag in CD3-TCR signaling in the thymus. In this report we assessed the constitutive interactions of CD5 in freshly isolated human thymocytes with signaling elements of the CD3-TCR complex. We determined that the ZAP-70 protein tyrosine kinase was present in CD5 immunoprecipitates. The two molecules were constitutively tyrosine phosphorylated in a complex also associating with unphosphorylated as well as phosphorylated zeta-chains. Although both p21 and p23 tyrosine-phosphorylated forms of zeta as well as phospho-CD3 epsilon molecules were constitutively present in human thymocytes and could be immunoprecipitated with ZAP-70- or CD3 epsilon-specific Abs, the p21 species of zeta was predomi...

Cell Death and Disease, 2014

Accurate regulation of nuclear factor-jB (NF-jB) activity is crucial to prevent a variety of diso... more Accurate regulation of nuclear factor-jB (NF-jB) activity is crucial to prevent a variety of disorders including immune and inflammatory diseases. Active NF-jB promotes IjBa and A20 expression, important negative regulatory molecules that control the NF-jB response. In this study, using two-hybrid screening we identify the RING-type zinc-finger protein 114 (RNF114) as an A20-interacting factor. RNF114 interacts with A20 in T cells and modulates A20 ubiquitylation. RNF114 acts as negative regulator of NF-jB-dependent transcription, not only by stabilizing the A20 protein but also IjBa. Importantly, we demonstrate that in T cells, the effect of RNF114 is linked to the modulation of T-cell activation and apoptosis but is independent of cell cycle regulation. Altogether, our data indicate that RNF114 is a new partner of A2O involved in the regulation of NF-jB activity that contributes to the control of signaling pathways modulating T cell-mediated immune response.

médecine/sciences, 2006

T cell clonal expansion contributing to host defense against pathogens is a tightly controlled pr... more T cell clonal expansion contributing to host defense against pathogens is a tightly controlled process to maintain the homeostasis of the immune system. Our understanding of how T cell growth and proliferation are controlled following antigenic stimulation is therefore a major challenge. Antigen recognition occurs when a naive T lymphocyte contacts an antigen-presenting cell. A specialized junction enriched in T-cell receptors, costimulation molecules and signaling adaptors, called the immunological synapse, is then created for several hours between the two cell types. Recent discoveries now clarify the molecular mechanisms used by this organization to control T cell growth and proliferation. It has been established that the immunological synapse functions in fact as an integrative platform where class Ia phosphoinositide-3-kinases (PI3Ks) are recruited and activated to continuously produce high levels of 3&amp;amp;amp;amp;amp;amp;amp;#39;-phosphoinositides. These lipids regulate the localization and the activation of a wide range of PH-domain containing proteins, among which the serine-threonine kinase Akt, a downstream effector of PI3Ks, appears to be a key player. FoxO (Forkhead subgroup O) family members control in various cell systems genes implicated in apoptosis, stress resistance and cell cycle arrest, thereby contributing to maintain quiescence in unstimulated cells. In naïve T cells contacting antigen-presenting cells a rapid but also very prolonged nuclear exclusion of these transcription factors is observed downstream of Akt. Mainly, this compartmentalization process is mandatory to induce T cell growth triggered by the T cell/antigen-presenting cell interaction. These findings demonstrate that to initiate cell cycle progression the formation of the immunological synapse is an undemanding tactic used by primary T cells to securely maintain the 3&amp;amp;amp;amp;amp;amp;amp;#39;-phosphoinositide-dependent mitotic switch governed by the spatial control of FoxO transcription factors.

Molecular and Cellular Biology, 2004

Biochemistry research international, 2012

SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques ... more SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques have been used to explore the functional consequences of protein SUMOylation. Most approaches consider the identification of sequences on substrates, adaptors, or receptors regulating the SUMO conjugation, recognition, or deconjugation. The large majority of the studied SUMOylated proteins contain the sequence [IVL]KxE. SUMOylated proteins are recognized by at least 3 types of hydrophobic SUMO-interacting motifs (SIMs) that contribute to coordinate SUMO-dependent functions. Typically, SIMs are constituted by a hydrophobic core flanked by one or two clusters of negatively charged amino acid residues. Multiple SIMs can integrate SUMO binding domains (SBDs), optimizing binding, and control over SUMO-dependent processes. Here, we present a survey of the methodologies used to study SUMO-regulated functions and provide guidelines for the identification of cis and trans sequences controlling SU...

Scientific reports, 2013

SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse ... more SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse cellular responses. Here SIMs were used to develop SUMO-traps to capture endogenous SUMOylated proteins. Our results show that these small peptides are transferable motifs that maintain their SUMO binding capacity when fused to the heterologous carrier protein GST. The tandem disposition of SIMs increases the binding capacity of SUMO-traps to specifically interact with polySUMO but not poly-Ubiquitin chains. We demonstrate that this SUMO capturing system purifies SUMOylated proteins such as IκBα, PTEN, PML or p53 in vitro and in vivo. These properties can be used to explore the many critical functions regulated by protein SUMOylation.

Methods, 2014

The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PT... more The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is tightly controlled by post-translational modifications (PTMs) including ubiquitin or Small Ubiquitin-related MOdifiers (SUMO). It is known that SUMOylation by SUMO-1, SUMO-2/-3, mono-or polyubiquitylation have a distinct impact on PTEN activity, localisation and/or stability, however the molecular mechanisms governing these processes are still unclear. Studying PTM regulated events has always been a difficult task due to their labile nature. Here, we propose an update on the role of these PTMs on PTEN function, as well as a methodological overview on the use of molecular traps named SUMO Binding Entities (SUBEs) or Tandem Ubiquitin Binding Entities (TUBEs) to capture SUMOylated or Ubiquitylated forms of PTEN respectively. When combined with in vitro SUMOylation or Ubiquitylation assays, the use of molecular traps facilitate the detection of modified forms of PTEN. SUMO and ubiquitin-traps are also suitable to capture endogenously modified forms of PTEN after expression of E3 ligases or treatment with chemical inhibitors. This versatile approach represents an interesting alternative to explore PTEN regulation by SUMO and ubiquitin under physiological or pathological conditions.

Methods in Molecular Biology, 2012

Studying postubiquitylation events has always been a diffi cult task due to the labile nature of ... more Studying postubiquitylation events has always been a diffi cult task due to the labile nature of these posttranslational modifi cations. When utilized in tandem, ubiquitin-binding entities (TUBEs) not only increase up to thousand times the affi nity for poly-ubiquitin chains but also protect ubiquitylated proteins from the action of the proteasome and de-ubiquitylating enzymes.

Molecular Oncology, 2014

The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, se... more The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2-dependent ubiquitylation and 26S

The FASEB Journal, 2001

The mechanisms by which caspases are stimulated following T cell receptor engagement are presentl... more The mechanisms by which caspases are stimulated following T cell receptor engagement are presently unknown. We show here that TCR cross-linking induces caspase activation, annexin V binding, loss of cell viability, Fyn cleavage, and apoptosis in a T cell hybridoma. All these events are increased in T cells overexpressing wild-type Fyn and are inhibited in cells expressing a kinase dead mutant or a soluble form of Fyn. Moreover, DNA fragmentation mediated by various proapoptotic stimuli and anti-CD3-induced caspase activation and DNA fragmentation are reduced significantly in mouse embryo fibroblasts and thymocytes from Fyn knockout mice respectively. These results indicate that an active and membrane-anchored Fyn is required for T cell receptor-induced caspase activation and apoptosis in T lymphocytes and point out a specific role of Fyn in caspase activation and apoptosis in T cells.

Nano Letters, 2009

Controlled nanoscale self-assembly of magnetic entities in semiconductors opens novel perspective... more Controlled nanoscale self-assembly of magnetic entities in semiconductors opens novel perspectives for the tailoring of magnetic semiconductor films and nanostructures with room temperature functionality. We report that a strongly directional self-assembly in growth direction in Mn-alloyed Ge is due to a stacking of individual Ge(1-x)Mn(x) clusters. The clusters represent the relevant entities for the magnetization of the material. They are formed of a core-shell structure displaying a Mn concentration gradient. While the magnetic moments seem to be carried by the shells of the clusters, their core is magnetically inactive.

Molecular and Cellular Biology, 2003

NF-B1 p105 functions both as a precursor of NF-B1 p50 and as a cytoplasmic inhibitor of NF-B. Fol... more NF-B1 p105 functions both as a precursor of NF-B1 p50 and as a cytoplasmic inhibitor of NF-B. Following the stimulation of cells with tumor necrosis factor alpha (TNF-␣), the IB kinase (IKK) complex rapidly phosphorylates NF-B1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-␣ to trigger p105 degradation, which releases the associated Rel/NF-B subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser 927 -Gly-Val-Glu-Thr-Ser 932 ) homologous to the IKK target sequence in IB␣. In this study, TNF-␣-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1 ؊/؊ and IKK2 ؊/؊ double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-␣ to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105 758-967 fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for ␤TrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of ␤TrCP by RNA interference blocks TNF-␣-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that ␤TrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, ␤TrCP affinity for the IKKphosphorylated sequence on p105 is substantially lower than that on IB␣. Thus, it appears that reduced p105 recruitment of ␤TrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-␣ stimulation relative to that for IB␣.