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Biomedical subjects

J L Rosa

Publications and source records attributed to J L Rosa.

At least 19 recordsLinked to original sources
PubMed2006

Repression of SOX6 transcriptional activity by SUMO modification.

SOX6 plays key functions in several developmental processes, including neurogenesis and skeleton formation. In this report, we show that SOX6 is modified in vitro and in vivo by small ubiquitin-related modifier (SUMO) on two distinct sites. Mutation of both sites abolished SOX6 sumoylation and increased SOX6 transcriptional activity. SUMO dependent repression of SOX6 transcription was promoted by UBC9 whereas siRNA to UBC9, cotransfection of inactive UBC9 or a SUMO protease increased SOX6 transcriptional activity. Furthermore, co-expression of SOX6 with SUMO2 results in the appearance of SOX6 in a punctate nuclear pattern that colocalized with promyelocytic leukemia protein, which was partially abolished by mutations in SOX6 sumoylation sites.

Animals↗
PubMed2005

The HERC proteins: functional and evolutionary insights.

HERC proteins are defined as containing both HECT and RCC1-like domains in their amino acid sequences. Six HERC genes have turned up in the human genome which encode two different sorts of polypeptides: while the small HERC proteins possess little more than the two aforementioned domains, the large ones are giant proteins with a plethora of potentially important regions. It is now almost 10 years since the discovery of the first family member and information is starting to accumulate pointing to a general role for these proteins as ubiquitin ligases involved in membrane-trafficking events. In this review, the available data on these six members are discussed, together with an account of their evolution.

Evolution, Molecular↗
PubMed2004

Abnormal alpha-synuclein interactions with rab3a and rabphilin in diffuse Lewy body disease.

The present study examines alpha-synuclein interactions with rab3a and rabphilin by antibody arrays, immunoprecipitation and pull-down methods in the entorhinal cortex of control cases and in diffuse Lewy body disease (LBD) cases. Alpha-synuclein immunoprecipitation revealed alpha-synuclein binding to rabphilin in control but not in LB cases. Immunoprecipitation with rab3a disclosed rab3a binding to rabphilin in control but not in LB cases. Moreover, rab3a interacted with high molecular weight (66 kDa) alpha-synuclein only in LB cases, in agreement with parallel studies using antibody arrays. Results were compared with pull-down assays using His(6)/Flag-tagged rab3, rab5 and rab8, and anti-Flag immunoblotting. Weak bands of 17 kDa, corresponding to alpha-synuclein, were obtained in LB and, less intensely, in control cases. In addition, alpha-synuclein-immunoreactive bands of high molecular weight (36 kDa) were seen only in LB cases after pull-down assays with rab3a, rab5 or rab8. These findings corroborate previous observations showing rab3a-rabphilin interactions in control brains, and add substantial information regarding decreased binding of rab3a to rabphilin and increased binding of rab3a to alpha-synuclein aggregates in LB cases. Since, alpha-synuclein, rab3a and rabphilin participate in the docking and fusion of synaptic vesicles, it can be suggested that exocytosis of neurotransmitters may be impaired in LB diseases.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Abnormal alpha-synuclein interactions with Rab proteins in alpha-synuclein A30P transgenic mice.

Mutation A30P in the alpha-synuclein gene is a cause of familial Parkinson disease. Transgenic mice expressing wild mouse and mutant human A30P alpha-synuclein, Tg5093 mice (Tg), show a progressive motor disorder characterized by tremor, rigidity, and dystonia, accompanied by accumulation of alpha-synuclein in the soma and neurites and by a conspicuous gliosis beginning in the hippocampal formation at the age of 7 to 8 months and spreading throughout the CNS. Impaired short-term changes in synaptic strength have also been documented in hippocampal slices from Tg mice. Alpha-synuclein aggregates of approximately 34 and 70 kDa, in addition to the band of 17 kDa, corresponding to the molecular weight of alpha-synuclein, were recovered in the PBS-soluble fraction of brain homogenates from Tg mice but not from brain samples from age-matched wildtype littermates. MPTP-treated Tg and wildtype mice produced alpha-synuclein aggregates in the PBS-, deoxycholate-, and SDS-soluble fractions. Aggregates of alpha-synuclein, although with different molecular weights, were also observed in rotenone-treated Tg and wildtype mice. Pull-down studies with members of the Rab protein family have shown that alpha-synuclein from Tg mice interacts with Rab3a, Rab5, and Rab8. This binding is not due to the amount of alpha-synuclein (levels of which are higher in Tg mice) and it is not dependent on the amount of Rab protein used in the assay. Rather, alpha-synuclein interactions with Rab proteins are due to mutant alpha-synuclein as demonstrated in Rab pull-down assays with recombinant of wildtype and mutant A30P human alpha-synuclein. Since Rab3a, Rab5, and Rab8 are important proteins involved in synaptic vesicle trafficking and exocytosis at the synapse, vesicle endocytosis, and trans-Golgi transport, respectively, it can be suggested that these functions are impaired in Tg mice. This rationale is consistent with previous data showing that short-term hippocampal synaptic plasticity is altered and that alpha-synuclein accumulates in the cytoplasm of neurons in Tg mice.

Animals↗
PubMed2003

Induction of the Sry-related factor SOX6 contributes to bone morphogenetic protein-2-induced chondroblastic differentiation of C3H10T1/2 cells.

Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis.

Animals↗
PubMed2001

The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3): promoter characterization and genomic structure.

A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.

1-Methyl-3-isobutylxanthine↗
PubMed2001

HERC3 binding to and regulation by ubiquitin.

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.

Animals↗
PubMed2000

Interaction and functional cooperation of NF-kappa B with Smads. Transcriptional regulation of the junB promoter.

The transforming growth factor-beta (TGF-beta) family of cytokines regulates diverse cellular processes through control of the expression of target genes. Smad proteins are a recently identified family of signal transducers for members of the TGF-beta family. Smads act as transcriptional regulators through binding to DNA and interacting with a variety of transcription factors. Here, we identified a kappaB site as a TGF-beta-responsive region in the 3'-downstream junB promoter region. We also demonstrate that kappaB sites alone are sufficient to mediate immediate transcriptional activation by TGF-beta. Transactivation of kappaB sites by TGF-beta requires an intact NF-kappaB pathway, cooperates with known activators of this pathway, and is mediated by Smad family members. Furthermore, we show that Smad3 interacts with p52 in vivo. These data expand the model in which Smad proteins undergo multiple interactions with several transcription factors that could induce either activation or repression of gene expression.

Animals↗
PubMed2000

Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to golgi membranes.

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membrane-bound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RIIalpha as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of beta-COP onto Golgi membranes containing prebound ARF1.

ADP-Ribosylation Factor 1↗
PubMed2000

Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression.

The ability to overexpress 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase (PFK-2)/(FBPase-2) or a truncated form of the enzyme with only the bisphosphatase domain allowed us to analyze the relative role of the kinase and the bisphosphatase activities in regulating fructose 2,6-bisphosphate (Fru-2,6-P(2)) concentration and to elucidate their differential metabolic impact in epithelial Mv1Lu cells. The effect of overexpressing PFK-2/FBPase-2 resulted in a small increase in the kinase activity and in the activity ratio of the bifunctional enzyme, increasing Fru-2,6-P(2) levels, but these changes had no major effects on cell metabolism. In contrast, expression of the bisphosphatase domain increased the bisphosphatase activity, producing a significant decrease in Fru-2,6-P(2) concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well as a delay in cell cycle. These results provide support for Fru-2,6-P(2) as a regulator of glycolytic flux and point out the role of glycolysis in cell cycle progression.

Adenosine Triphosphate↗
PubMed1999

A zinc-finger transcription factor induced by TGF-beta promotes apoptotic cell death in epithelial Mv1Lu cells.

Transforming growth factor-beta (TGF-beta) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc-finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF-beta in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF-beta effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl-2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co-expression of Bcl-2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro-apoptotic properties of TGF-beta.

Animals↗
PubMed1998

Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2.

We show here that Vav-2, a member of the Vav family of oncoproteins, acts as a guanosine nucleotide exchange factor (GEF) for RhoG and RhoA-like GTPases in a phosphotyrosine-dependent manner. Moreover, we show that Vav-2 oncogenic activation correlates with the acquisition of phosphorylation-independent exchange activity. In vivo, wild-type Vav-2 is activated oncogenically by tyrosine kinases, an effect enhanced further by co-expression of RhoA. Likewise, the Vav-2 oncoprotein synergizes with RhoA and RhoB proteins in cellular transformation. Transient transfection assays in NIH-3T3 cells show that phosphorylated wild-type Vav-2 and the Vav-2 oncoprotein induce cytoskeletal changes resembling those observed by the activation of the RhoG pathway. In contrast, the constitutive expression of the Vav-2 oncoprotein in rodent fibroblasts leads to major alterations in cell morphology and to highly enlarged cells in which karyokinesis and cytokinesis frequently are uncoupled. These results identify a regulated GEF for the RhoA subfamily, provide a biochemical explanation for vav family oncogenicity, and establish a new signaling model in which specific Vav-like proteins couple tyrosine kinase signals with the activation of distinct subsets of the Rho/Rac family of GTPases.

3T3 Cells↗
PubMed1998

Characterization of human hect domain family members and their interaction with UbcH5 and UbcH7.

The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of E6-AP, an E3 ubiquitin-protein ligase. Since the C terminus of E6-AP mediates thioester complex formation with ubiquitin, a necessary intermediate step in E6-AP-dependent ubiquitination, it was proposed that members of the hect domain family in general have E3 activity. The hect domain is approximately 350 amino acids in length, and we show here that the hect domain of E6-AP is necessary and sufficient for ubiquitin thioester adduct formation. Furthermore, the human genome encodes at least 20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of E3s, several of these are shown to form thioester complexes with ubiquitin. In addition, some hect domain proteins interact preferentially with UbcH5, whereas others interact with UbcH7, indicating that human hect domain proteins can be grouped into at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of ubiquitin-dependent proteolytic pathways.

Amino Acid Sequence↗
PubMed1998

JunB is involved in the inhibition of myogenic differentiation by bone morphogenetic protein-2.

Bone morphogenetic proteins (BMPs) constitute a family of multifunctional growth and differentiation factors structurally related to transforming growth factor-beta. BMPs were first identified by their osteoinductive effects, inducing ectopic bone formation when implanted in skeletal muscle, and have an important role as regulators of skeletal development in vivo. In vitro, BMP-2 is able to transdifferentiate myogenic C2C12 cells into the osteoblastic phenotype. In this report, we show that the osteoinductive effects of BMP-2 in C2C12 cells are mediated by bone morphogenetic protein receptor type-IA in combination with both activin receptor type II and bone morphogenetic protein receptor type II. We also analyzed the expression levels of nuclear protooncogenes to understand early transcriptional events induced by BMP-2. We show that junB is an immediate early gene induced by BMP-2 and transforming growth factor-beta. BMP-2 induces transcriptional activation of JunB expression as early as 30 min after ligand addition, reaching maximal levels after 90 min. Increase of JunB mRNA correlates with a higher AP-1 binding activity. Furthermore, ectopic overexpression of JunB is sufficient to inhibit expression of myoblast differentiation markers in C2C12 cells. These data, taken together, show the involvement of JunB in the early steps of inhibition of myogenic differentiation induced by transforming growth factor-beta family members.

Bone Morphogenetic Protein 2↗
PubMed1998

Molecular cloning, expression, and chromosomal localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/ fructose-2, 6-bisphosphatase gene (PFKFB3).

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15-->p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.

Animals↗
PubMed1997

A giant protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins forms a cytosolic ternary complex with clathrin and Hsp70.

We have recently identified an unusually large human protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins (EMBO J 15: 4262-4273, 1996). This protein, designated p532 based on its predicted molecular weight (EMBO J 15: 5738, 1996), contains multiple structural domains including two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP-binding protein Ran, seven beta-repeat domains characteristic of the beta subunit of heterotrimeric G proteins, three putative SH3 binding sites, a putative leucine-zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. Some of these domains are known to be involved in protein-protein interactions, suggesting the existence of p532-interacting proteins. To identify some of these proteins, we used the carboxy-terminal RCC1-like domain (RLD) of p532 in the yeast two-hybrid system. We report here the isolation of a clone that encodes the last 654 amino acid residues of the clathrin heavy chain. This interaction involves amino acid residues 1315-1557 of the clathrin heavy chain and the carboxy, but not the amino-terminal RLD of p532. p532 has been located in the cytosolic fraction as well as in the Golgi apparatus. The interaction between p532 and clathrin only occurs in the cytosol and is mediated by the formation of an ATP-dependent ternary complex with the heat shock protein, Hsp70. These observations suggest that p532 is involved in membrane transport processes.

ADP-Ribosylation Factor 1↗