Biomedical subjects

G Pedraza-Alva

Publications and source records attributed to G Pedraza-Alva.

7 recordsLinked to original sources

PubMed2001

Regulation of Cbl molecular interactions by the co-receptor molecule CD43 in human T cells.

CD43, one of the most abundant glycoproteins on the T cell surface, has been implicated in selection and maturation of thymocytes and migration, adhesion, and activation of mature T cells. The adapter molecule Cbl has been shown to be a negative regulator of Ras. Furthermore, it may also regulate intracellular signaling through the formation of several multi-molecular complexes. Here we investigated the role of Cbl in the CD43-mediated signaling pathway in human T cells. Unlike T cell receptor signaling, the interaction of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase, resulting from CD43-specific signals, is independent of Cbl tyrosine phosphorylation, suggesting an alternative mechanism of interaction. CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the tau-isoform of 14-3-3. protein. Protein kinase C-mediated Cbl serine phosphorylation was required for this interaction, because the PKC inhibitor RO-31-8220 prevented it, as well as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the CD43-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and AP-1 transcriptional activity, confirming nevertheless a negative role for Cbl in T cell signaling. However, under normal conditions, PKC activation resulting from CD43 engagement was required to activate the MAPK pathway, suggesting that phosphorylation of Cbl on serine residues by PKC and its association with 14-3-3 molecules may play a role in preventing the Cbl inhibitory effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate the molecular associations and functions of the Cbl adapter protein.

14-3-3 Proteins↗

PubMed2000

CD43-mediated signals induce DNA binding activity of AP-1, NF-AT, and NFkappa B transcription factors in human T lymphocytes.

Although numerous reports document a role for CD43 in T cell signaling, the direct participation of this molecule in cell activation has been questioned. In this study we show that CD43 ligation on human normal peripheral T cells was sufficient to induce interleukin-2, CD69, and CD40-L gene expression, without requiring signals provided by additional receptor molecules. This response was partially inhibited by cyclosporin A and staurosporine, suggesting the participation of both the Ca(2+) and the protein kinase C pathways in CD43 signaling. Consistent with the transient CD43-dependent intracellular Ca(2+) peaks reported by others, signals generated through the CD43 molecule resulted in the induction of NF-AT DNA binding activity. CD43-dependent signals resulted also in AP-1 and NFkappaB activation, probably as a result of protein kinase C involvement. AP-1 complexes bound to the AP-1 sequence contained c-Jun, and those bound to the NF-AT-AP-1 composite site contained c-Jun and Fos. NFkappaB complexes containing p65 could be found as early as 1 h after CD43 cross-linking, suggesting that CD43 participates in early events of T cell activation. The induction of the interleukin-2, CD69, and CD-40L genes and the participation of AP-1, NF-AT, and NFkappaB in the CD43-mediated signaling cascade implicate an important role for this molecule in the regulation of gene expression and cell function.

Antigens, CD↗

PubMed1999

CD43, a molecule with multiple functions.

To initiate a specific immune response, lymphoid cells integrate a variety of signals generated through the orchestrated interaction of multiple cell surface molecules with their counter-receptors. As a result of the specific recognition of the antigen through antigen-specific receptors, and of the monitoring of their particular environment through the so-called coreceptor molecules, lymphoid cells go through elaborate processes of maturation and activation, contributing to the plasticity and sensitivity of immune response. CD43 is the major sialic acid rich protein on the surface of lymphocytes. However, the specific roles of this protein in different lymphoid cells under normal physiological conditions remain largely unknown. In this review we will mainly focus on the recent advances concerning the functions of this molecule as a coreceptor of different lymphoid cells as well as on the participation of this molecule in different pathologies.

Animals↗

PubMed1998

T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation.

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signals that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in signal transduction pathway of the CD43 molecule are only beginning to be understood. We have shown recently that cross-linking CD43 on the cell surface of human T lymphocytes with the anti-CD43 monoclonal antibody L10 leads to CD43-Fyn kinase interactions and to Fyn phosphorylation on tyrosine residues. This interaction seems to be mediated by the SH3 domain of Fyn and a proline-rich sequence located in the cytoplasmic domain of CD43. Here we show that CD43-specific activation of human T lymphocytes induced tyrosine phosphorylation of the adaptor protein Shc and of the guanine exchange factor Vav, as well as the formation of a macromolecular complex that comprises Shc, GRB2, and Vav. CD43 ligation resulted in enhanced formation of Vav.SLP-76 complexes and in the activation and nuclear translocation of ERK2. Cross-linking of the CD43 molecule in 3T3-CD43(+) cells induced luciferase activity from a construct under the control of the Fos serum responsive element. Altogether, these data suggest that the mitogen-activated protein kinase pathway is involved in CD43-dependent interleukin-2 gene expression.

3T3 Cells↗

PubMed1996

CD43-specific activation of T cells induces association of CD43 to Fyn kinase.

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signal transduction pathways that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in CD43's signal transduction pathway are poorly understood. In the present report we show that activation of both purified T lymphocytes and Jurkat cells, through CD43 cross-linking with the anti-CD43 L10 monoclonal antibody, induced CD43 association to Fyn kinase. This association is mediated by the Src homology 3 (SH3) domain of Fyn, since a glutathione S-transferase-Fyn SH3 fusion protein was able to precipitate CD43 from lysates of CD43-activated T cells. A synthetic peptide containing the SH3 binding sites of p85, located within the amino acid sequence 300ERQPAPALPPKPPKP314, was able to inhibit binding of CD43 to Fyn as well as to the glutathione S-transferase-Fyn SH3 fusion protein. We also provide evidence that upon CD43 cross-linking, Fyn is tyrosine-phosphorylated in a time-dependent manner. Our results suggest that CD43 cross-linking on the T cell surface induces the interaction between CD43 and Fyn, presumably through the Fyn SH3 domain and a putative SH3 binding site in CD43, leading to Fyn tyrosine phosphorylation and signal propagation.

Amino Acid Sequence↗

PubMed1994

MyoD1 promoter autoregulation is mediated by two proximal E-boxes.

We show that in mouse myoblasts the MyoD1 promoter is highly stimulated by MyoD1 expression, suggesting that it is controlled by a positive feedback loop. Using deletion and mutation analyses, we identified the targets for MyoD1 promoter autoregulation as the two proximal E-boxes located close to the MyoD1 core promoter. Gel mobility shift competition assays with MyoD1 antibodies as competitor suggest that the MyoD1 protein is binding directly to these E-boxes. Autoregulation did not occur in fibroblasts cotransfected with the expression vector of MyoD1. It is assumed that autoregulation is controlled by the stoichiometry between the MyoD1 protein and negatively regulatory proteins like Id, which is known to be highly expressed in fibroblasts. When the MyoD1 promoter was methylated, autoregulation only occurred when the density of methylated sites was low. The density of DNA methylation, therefore, can determine the accessibility of the MyoD1 promoter to transcription factors and interfere with the auto- and crossregulatory loop. The MyoD1 promoter in vivo was found to be only partially methylated in all tissues tested except in skeletal muscle where it was demethylated. We propose that high level expression of the MyoD1 gene is a result of release from constraints such as negative regulatory factors and/or DNA methylation interfering with MyoD1 autoregulation.

Animals↗

PubMed1994

AP-1 binds to a putative cAMP response element of the MyoD1 promoter and negatively modulates MyoD1 expression in dividing myoblasts.

We have studied the transcriptional activity of the mouse MyoD1 gene promoter in vivo and in vitro using mouse G8 myoblasts and muscle cell nuclear extracts. 5' deletion analysis of the promoter and transcription-competition analysis using oligonucleotides corresponding to several cis-acting elements revealed that the basal activity of the MyoD1 promoter is conferred by two SP1 boxes, an AP-2 box, and a CAAT box. We have identified a negative regulatory sequence located between nucleotide position -342 to -322 with respect to the cap site. The negative regulatory element shows sequence homology with cAMP-responsive element (CRE) and AP-1 binding site (5'-GAGCACTGAGGTCAGTACAG-3'). As determined by gel mobility shift competition analysis, oligonucleotides containing AP-1 binding sites inhibit protein interactions with the MyoD1 CRE-like element. We also show that binding to this element is down-regulated during myogenic differentiation and can be reinduced by the addition of serum. Furthermore, mutation of the CRE-like element induces MyoD promoter activity in diving myoblasts. By using anti-c-Fos antibodies we show that AP-1 is binding to the MyoD1 CRE-like element. Our results indicate that AP-1 negatively modulates MyoD1 expression in growing myoblasts and strongly suggest that c-Fos and c-Jun inhibit myogenesis and MyoD1 expression by direct binding to a negative cis-acting element in the MyoD1 promoter.

Animals↗