Biomedical subjects

J W Ramos

Publications and source records attributed to J W Ramos.

7 recordsLinked to original sources

PubMed2001

Identification of cell signaling molecules by expression cloning.

Using expression cloning one can isolate proteins with specific biological functions. This methodology can be adapted for the identification of novel players in the regulation of cell signaling. Here, we describe an expression cloning strategy to identify suppressors of Ras signaling. This screen is based on the observation that the activation of the small guanosine triphosphate (GTP)-binding protein H-Ras initiates a mitogen-activated protein kinase (MAPK)-dependent signaling pathway that inactivates integrin ligand binding. Our strategy depends on flow cytometry and a monoclonal antibody that recognizes integrin activation states. Flow cytometry enhances the screen's sensitivity thereby allowing us to examine function quantitatively at the level of a single cell millions of times in one screen. The following protocol provides a detailed method for the isolation of proteins that regulate cell signaling.

Animals↗

PubMed2001

PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

3T3 Cells↗

PubMed2000

Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism.

PEA-15 is a small, death effector-domain (DED)-containing protein that was recently demonstrated to inhibit tumor necrosis factor-alpha-induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5'-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.

3T3 Cells↗

PubMed1998

The death effector domain of PEA-15 is involved in its regulation of integrin activation.

Increased integrin ligand binding affinity (activation) is triggered by intracellular signaling events. A Ras-initiated mitogen-activated protein kinase pathway suppresses integrin activation in fibroblasts. We used expression cloning to isolate cDNAs that prevent Ras suppression of integrin activation. Here, we report that PEA-15, a small death effector domain (DED)-containing protein, blocks Ras suppression. PEA-15 does not block the capacity of Ras to activate the ERK mitogen-activated protein kinase pathway. Instead, it inhibits suppression via a pathway blocked by a dominant-negative form of the distinct small GTPase, R-Ras. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to reverse suppression of integrin activation. Thus, certain DED-containing proteins can regulate integrin activation as opposed to apoptotic protease cascades.

Amino Acid Sequence↗

PubMed1997

Complementation of dominant suppression implicates CD98 in integrin activation.

The integrin family of adhesion receptors are involved in cell growth, migration and tumour metastasis. Integrins are heterodimeric proteins composed of an alpha and a beta subunit, each with a large extracellular, a single transmembrane, and a short cytoplasmic domain. The dynamic regulation of integrin affinity for ligands in response to cellular signals is central to integrin function. This process is energy dependent and is mediated through integrin cytoplasmic domains. However, the cellular machinery regulating integrin affinity remains poorly understood. Here we describe a genetic strategy to disentangle integrin signalling pathways. Dominant suppression occurs when overexpression of isolated integrin beta1 cytoplasmic domains blocks integrin activation. Proteins involved in integrin signalling were identified by their capacity to complement dominant suppression in an expression cloning scheme. CD98, an early T-cell activation antigen that associates with functional integrins, was found to regulate integrin activation. Furthermore, antibody-mediated crosslinking of CD98 stimulated beta1 integrin-dependent cell adhesion. These data indicate that CD98 is involved in regulating integrin affinity, and validate an unbiased genetic approach to analysing integrin signalling pathways.

Animals↗

PubMed1996

Xenopus embryonic cell adhesion to fibronectin: position-specific activation of RGD/synergy site-dependent migratory behavior at gastrulation.

During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg-Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.

Activins↗

PubMed1996

Integrin-dependent adhesive activity is spatially controlled by inductive signals at gastrulation.

Integrins mediate cell-ECM interactions essential for morphogenesis, however, the extent to which integrin adhesive activities are regulated in the embryo has not been addressed. We report that integrin-dependent cell adhesion to the Arg-Gly-Asp (RGD) containing central cell-binding domain of fibronectin is required for gastrulation in Xenopus. Although all cells of the early embryo retain the ability to attach to this region, only involuting cells arising from the dorsal and ventral lips of the blastopore are able to spread and migrate on fibronectin in vitro. This change in adhesive behavior is mimicked by treating animal cap cells with activin-A. Activin-induced changes in adhesion are independent of new transcription, translation, or changes in receptor expression at the cell surface. We demonstrate that ectopic expression of integrin alpha4beta1 in animal cap cells results in attachment to the non RGD-containing V-region of fibronectin. Further, these cells acquire the ability to spread on the V-region following activin induction. Thus, alpha4beta1 adhesion to the V-region, like endogenous integrin binding to the central cell-binding domain, is responsive to activin signalling. These data indicate that cell adhesion to the central cell-binding domain is regulated in both space and time, and is under the control of inductive signals that initiate gastrulation movements. We suggest that position-specific inductive interactions are likely to represent a novel and general mechanism by which integrin adhesion is modulated throughout development.

Activins↗