Francis Vilbois

Phosphorylation of Bid by Casein Kinases I and II Regulates Its Cleavage by Caspase 8

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.

Proteomic Analysis of the Mouse Liver Mitochondrial Inner Membrane

Mitochondria play a crucial role in cellular homeostasis, which justifies the increasing interest in mapping the different components of these organelles. Here we have focused our study on the identification of proteins of the mitochondrial inner membrane (MIM). This membrane is of particular interest because, besides the well known components of the respiratory chain complexes, it contains several ion channels and many carrier proteins that certainly play a key role in mitochondrial function and, therefore, deserve to be identified at the molecular level. To achieve this goal we have used a novel approach combining the use of highly purified mouse liver mitochondrial inner membranes, extraction of membrane proteins with organic acid, and two-dimensional liquid chromatography coupled to tandem mass spectrometry. This procedure allowed us to identify 182 proteins that are involved in several biochemical processes, such as the electron transport machinery, the protein import machinery, protein synthesis, lipid metabolism, and ion or substrate transport. The full range of isoelectric point (3.9–12.5), molecular mass (6–527 kDa), and hydrophobicity values (up to 16 transmembrane predicted domains) were represented. In addition, of the 182 proteins found, 20 were unknown or had never previously been associated with the MIM. Overexpression of some of these proteins in mammalian cells confirmed their mitochondrial localization and resulted in severe remodeling of the mitochondrial network. This study provides the first proteome of the MIM and provides a basis for a more detailed study of the newly characterized proteins of this membrane.

The stress-induced MAP kinase p38 regulates endocytic trafficking via the GDI : Rab5 complex

Early endocytic membrane traffic is regulated by the small GTPase Rab5, which cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle depends on GDI, which functions as a Rab vehicle in the aqueous environment of the cytosol. Here, we report that formation of the GDI:Rab5 complex is stimulated by a cytosolic factor that we purified and then identified as p38 MAPK. We find that p38 regulates GDI in the cytosolic cycle of Rab5 and modulates endocytosis in vivo. Our observations reveal the existence of a cross-talk between endocytosis and the p38-dependent stress response, thus providing molecular evidence that endocytosis can be regulated by the environment.

Differential sorting and fate of endocytosed GPI-anchored proteins

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI‐ APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI‐APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft‐like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI‐APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI‐APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI‐APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI‐APs in different cell types depend on the residence time of GPI‐APs in lipid rafts, and hence that raft partitioning regulates GPI‐APs sorting in the endocytic pathway.

Leukocyte transmigration is modulated by chemokine-mediated PI3Kγ-dependent phosphorylation of vimentin

Phosphoinositide 3‐kinase γ (PI3Kγ) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine‐stimulated WT or PI3Kγ‐null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kγ signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kγ or the kinase activity of PI3Kγ (PI3KγKD/KD), phosphorylation of vimentin was reduced. PI3Kγ‐null macrophages displayed impaired chemokine‐driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N‐terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kγ‐null macrophages with a vimentin mutant mimicking serine phosphorylation of N‐terminal residues rescued the transendothelial migration defect. These results define vimentin N‐terminal phosphorylation and fiber reorganization as a target of chemokine‐dependent PI3Kγ signaling in leukocytes.

A Chemical Proteomics Approach to Phosphatidylinositol 3-Kinase Signaling in Macrophages

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid “baits” with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.

c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-β (SAPKβ) binds and phosphorylates the neuronal microtubule regulator SCG10

The neuronal growth‐associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule‐destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine–threonine‐directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen‐activated protein (MAP) kinases, c‐Jun N‐terminal/stress‐activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKβ. JNK3/SAPKβ phosphorylation occurs at Ser‐62 and Ser‐73, residues that result in reduced microtubule‐destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKβ activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)‐anchored proteins y two‐dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent‐resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI‐anchored proteins. Although the GPI‐anchored proteins were readily separated by one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), these proteins were undetectable on two‐dimensional (2‐D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent‐resistant membranes such as caveolin‐1 and flotillin‐1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI‐anchored proteins required cleavage of the lipid tail with phosphatidylinositol‐specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI‐anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI‐anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N‐glycosylation. Pretreatment with N‐glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N‐linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray‐tandem mass spectrometry as illustrated for the GPI‐anchored protein, Thy‐1.

The Redox Sensor TXNL1 Plays a Regulatory Role in Fluid Phase Endocytosis

Background Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis. Methodology/Principal Findings When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5. Conclusions/Significance Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.

Mapping and identification of protein‐protein interactions by two‐dimensional far‐Western immunoblotting

Studies of protein‐protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so‐called “substrate trapping” mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680—1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome‐wide approach to probe for candidate substrates in cellular extracts that were separated by two‐dimensional (2‐D) gel electrophoresis and blotted onto membranes. Protein‐protein interactions were revealed by far‐Western immunoblotting and positive binding proteins were subsequently identified from silver‐stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radiolabeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein‐protein interactions.