LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation.

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.

Measles virus assembly within membrane rafts.

During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305-311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly.

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.

The related adhesion focal tyrosine kinase differentially phosphorylates p130Cas and the Cas-like protein, p105HEF1.

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following beta1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130(Cas) and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130(Cas) and HEF1, phosphorylation of p130(Cas), but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130(Cas) and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130(Cas) domains. RAFTK was able to phosphorylate the substrate domains of both p130(Cas) and HEF1, but only the C-terminal domain of p130(Cas). Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130(Cas). These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130(Cas).

Regulation of integrin-mediated p130(Cas) tyrosine phosphorylation in human B cells. A role for p59(Fyn) and SHP2.

Engagement of beta1 integrins in terminally differentiated human B cell lines, such as ARH-77, leads to prominent tyrosine phosphorylation of the p130 Crk-associated substrate (Cas). Cas regulates the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes, which are potentially involved in the propagation of downstream signals. We demonstrate here that immunoprecipitated Cas from beta1 integrin-stimulated ARH-77 cells was associated with tyrosine kinase and phosphatase activities and that integrin ligation led to the recruitment of at least p59(Fyn) tyrosine kinase and SHP2 tyrosine phosphatase in Cas immune complexes. Cotransfection studies in COS-7 cells further indicated that Fyn/Cas physical interaction and Fyn-mediated Cas phosphorylation required amino acids 638-889 in the C-terminal region of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding motifs. In vitro binding studies using glutathione S-transferase fusion proteins derived from the SH2 or SH3 domains of Fyn suggested that both Fyn domains can participate in Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential modulators of Cas signaling complexes in B cells.

Involvement of p130(Cas) and p105(HEF1), a novel Cas-like docking protein, in a cytoskeleton-dependent signaling pathway initiated by ligation of integrin or antigen receptor on human B cells.

The Crk-associated substrate p130(Cas) (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as "docking" molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either beta1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56(Lyn) immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of beta1 integrin or BCR on human B cells.

The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas.

Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKbeta, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for beta1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells.

Tyrosine phosphorylation of the product of the c-cbl protooncogene is [corrected] induced after integrin stimulation.

Integrin crosslinking on human B cells induces tyrosine phosphorylation of a set of proteins ranging from 105 to 130 kDa, among which is the focal adhesion kinase p125FAK. Here we show that the c-CBL protooncogene product p120c-CBL is a component of these substrates. beta 1 integrin stimulation of p120c-CBL phosphorylation was observed in both transformed and normal human B cells, and was inhibited by prior treatment of cells with cytochalasin B, which disrupts the actin network. In contrast, tyrosine phosphorylation of p120c-CBL following crosslinking of the B cell antigen receptor (BCR) was not affected by cytochalasin B. Integrin stimulation of the promegakaryocytic cell line MO7e also led to a cytoskeleton-dependent tyrosine phosphorylation of p120c-CBL. In MO7e cells, this stimulation was induced by ligation of either beta 1 or beta 2 integrin, whereas only by ligation of beta 1 integrin in B cells. Tyrosine phosphorylation of p120c-CBL links phosphatidylinositol-3 kinase (PI-3K) with the BCR signaling machinery. Although the p85 subunit of PI-3K was increased in p120c-CBL immunoprecipitates from BCR-stimulated B cells, this association was only minimally increased by beta 1 integrin ligation. The function of p120c-CBL remains unknown; however, its interactions in vitro and in vivo with Src homology 2 and 3 (SH2 and SH3) domain-containing proteins suggest that p120c-CBL has a significant function in signal transduction pathways, and therefore may play a role in integrin signaling in lymphoid and hematopoietic cells.

Association of the Cas-like molecule HEF1 with CrkL following integrin and antigen receptor signaling in human B-cells: potential relevance to neoplastic lymphohematopoietic cells.

CrkL, a cellular homologue of the v-crk oncogene, belongs to the family of adaptor proteins, containing SH2 and SH3 domains, but no catalytic domain. Stimulation of normal B-cells and B-cell lines through beta1 integrin or - cell antigen receptor (BCR) promoted the association of CrkL with a set of 105-130 kD tyrosine phosphorylated substrates. The principal substrate is a recently identified molecule known as p105HEF1 (HEF1), which is highly homologous to p130Cas (Cas), the major tyrosine-phosphorylated protein detected in fibroblasts after transformation by v-crk. Immunodepletion studies indicated that all the tyrosine phosphorylated HEF1 or Cas was complexed with CrkL. Furthermore, the guanine nucleotide exchange factor C3G, which is thought to be involved in the regulation of the ras pathway and constitutively binds to the C-terminal SH3 domain of CrkL, could be detected in HEF1 immunoprecipitates. Therefore, CrkL is involved in the formation of a HEF1-CrkL-C3G ternary complex in B-cells, suggesting that it is likely to play an important role, allowing the propagation of the stimulation initiated by both BCR and beta1 integrin ligation.

Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells.

B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.