Jose Luis Rosa

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.

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.

BMP-2 Induces Osterix Expression through Up-regulation of Dlx5 and Its Phosphorylation by p38

Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation.

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.

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 pO2 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-1α 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.

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.

Amino Acids Activate Mammalian Target of Rapamycin Complex 2 (mTORC2) via PI3K/Akt Signaling

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.

p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins

We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains 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. In addition, p619 possesses seven beta‐repeat domains characteristic of the beta‐subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid‐rich regions, a putative leucine zipper and a carboxy‐terminal HECT domain characteristic of E3 ubiquitin‐protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy‐terminal RCC1‐like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1‐like motif located at the amino‐terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R‐Ras2/TC21. These observations suggest that p619 is a Brefeldin A‐sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.

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-β. 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 thatjunB is an immediate early gene induced by BMP-2 and transforming growth factor-β. 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-β family members.

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

Transforming growth factor‐β (TGF‐β) 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‐β in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF‐β 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‐β.