Biomedical subjects

Nieves Ibarrola

Publications and source records attributed to Nieves Ibarrola.

6 recordsLinked to original sources

PubMed2004

Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network.

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

Animals↗

PubMed2004

A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine.

Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.

3T3-L1 Cells↗

PubMed2004

Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor beta signaling.

BACKGROUND: Transforming growth factor-betas (TGF-betas), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors. RESULTS: There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (associated molecule with the SH3 domain of STAM), an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in Jun kinase activation domain binding protein and proteasomal subunits) domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-beta-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-beta signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-beta. CONCLUSIONS: This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-beta signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-beta signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2003

A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture.

Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

Amino Acid Sequence↗

PubMed2003

Development of human protein reference database as an initial platform for approaching systems biology in humans.

Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.

BRCA1 Protein↗

PubMed2002

A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling.

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.

Adaptor Proteins, Signal Transducing↗