Biomedical subjects

Jose Luis Rosa

Publications and source records attributed to Jose Luis Rosa.

10 recordsLinked to original sources

PubMed2006

TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.

Amino Acid Sequence↗

PubMed2006

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel.

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.

Acrylamides↗

PubMed2005

Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins.

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P(2) bound to HERC1's RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P(2) bound to the RLD1.

ADP-Ribosylation Factor 6↗

PubMed2004

6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia.

The up-regulation of glycolysis to enhance the production of energy under reduced pO(2) is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5'-flanking region. Extensive analysis of the 5' pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1alpha null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.

Animals↗

PubMed2004

BMP-2 decreases Mash1 stability by increasing Id1 expression.

In neural development, bone morphogenetic proteins (BMPs) restrict neuronal differentiation, thereby promoting the maintenance of progenitor cells or even inducing astrocytogenesis. We report that exposure of neuroendocrine lung carcinoma cells to BMP-2 leads to a rapid decline in steady-state levels of Mash1 protein and some neuron-specific markers. BMP-2 induces a post-transcriptional decrease in Mash1 levels through enhanced degradation. We demonstrate that Mash1 protein stability is tightly regulated by the E47/Id1 expression ratio. Transient induction of Id1 by BMP-2 negatively correlates with Mash1 levels. Furthermore, an ectopic increase in Id1 levels is sufficient to induce degradation of either ectopic or endogenous Mash1, whereas expression of Mash1 in Id1-deficient cells or overexpression of E47 makes Mash1 levels refractory to the addition of BMP-2. Furthermore, we show that the E47/Id1 expression ratio also regulates CK2-mediated phosphorylation of Mash1 on Ser152, which increases interaction of Mash1-E47 heterodimers. We propose a novel mechanism in which the balance between Id and E protein levels regulates not only the transcriptional function but also protein stability of the neurogenic bHLH transcription factor Mash1.

Animals↗

PubMed2004

The giant protein HERC1 is recruited to aluminum fluoride-induced actin-rich surface protrusions in HeLa cells.

HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF(4)(-)) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF(4)(-), in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions.

ADP-Ribosylation Factor 6↗

PubMed2003

Regulation of ubiquitous 6-phosphofructo-2-kinase by the ubiquitin-proteasome proteolytic pathway during myogenic C2C12 cell differentiation.

6-Phosphofructo-2-kinase catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase. These properties confer to this bifunctional enzyme a key role in the control of glycolysis and gluconeogenesis. Several mammalian isozymes generated by alternative splicing from four genes, designated pfkfb1-4, have been identified. The results presented in this study demonstrate the expression of the pfkfb3 gene in C2C12 cells and its downregulation during myogenic cell differentiation. We also show that the decrease of ubiquitous 6-phosphofructo-2-kinase isozyme levels, product of pfkfb3 gene, is due to its enhanced degradation through the ubiquitin-proteasome proteolytic pathway.

Animals↗

PubMed2003

Interaction between HERC1 and M2-type pyruvate kinase.

HERC proteins are characterized by having one or more RCC1-like domains as well as a C-terminal HECT domain in their amino acid sequences. This has led researchers to suggest that they may act as both guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we describe a physical interaction between the HECT domain of HERC1, a giant protein involved in intracellular membrane traffic, and the M2 isoform of glycolytic enzyme pyruvate kinase (M2-PK). Partial colocalization of endogenous proteins was observed by immunofluorescence studies. This interaction neither induced M2-PK ubiquitination nor affected its enzymatic activity. The putative significance of the association is discussed.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Inhibition of PI3K/p70 S6K and p38 MAPK cascades increases osteoblastic differentiation induced by BMP-2.

Bone morphogenetic proteins (BMPs) transdifferentiate C2C12 cells from the myogenic to the osteogenic lineage. In this work we examine the role of the phosphatidylinositol 3-kinase/p70 S6 kinase (PI3K/p70 S6K) and p38 mitogen-activated protein kinase (p38 MAPK) cascades in the osteogenic effects of BMP-2. BMP-2 stimulated both cascades transiently (maximal at 1 h and decreasing thereafter). In contrast, BMP-2 had no effect on p42/p44 MAPK (Erks) stimulation. We also analyzed the effects of selective inhibitors of these pathways on the expression of osteogenic markers. Inhibitors of p38 MAPK (SB203580) or the PI3K/p70 S6K pathway (Ly294002 and rapamycin) not only fail to block the osteoblast phenotype induced by BMP-2, measured as induction of Cbfa1 expression and transcriptional activity, but also potentiate the effect of BMP-2 on late osteoblast markers, such as alkaline phosphatase activity and osteocalcin expression. These data suggest that, in contrast to their positive effect on myogenic differentiation, PI3K/p70 S6K and p38 MAPK cascades have a negative role in osteoblast differentiation.

Alkaline Phosphatase↗

PubMed2001

Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene.

Bone morphogenetic proteins (BMPs) are potent inhibitors of myoblast differentiation and inducers of bone formation both in vivo and in vitro. Expression of Id1, a negative regulator of basic helix-loop-helix transcription factors, is up-regulated by BMPs and contributes to the antimyogenic effects of this family of cytokines. In this report, we have identified a specific BMP-2 immediate early response enhancer in the human Id1 gene. Transcriptional activation of the enhancer was increased by overexpression of BMP-responsive Smads, and Smad4 and was completely abrogated in Smad4-deficient cells. Deletion analysis demonstrates that the responsive region is composed of two separate DNA binding elements, a set of overlapping GC boxes, which bind BMP-regulated Smads upon BMP stimulation, and three repeats of CAGAC boxes. Gel shift and oligonucleotide pull-down assays demonstrated that these two types of motifs were capable of binding their corresponding Smads. However, deletion or mutation of either DNA binding element was nonadditive, since disruption of either GC or CAGAC boxes resulted in complete or severe loss of BMP-2 responsiveness. These data suggest the simultaneous requirement of two independent DNA binding elements to allow functional cooperativity of BMP-regulated Smads and Smad4 in BMP-activated gene promoters.

Animals↗