Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis. (17/190738)

The Met tyrosine kinase - the HGF receptor - induces cell transformation and metastasis when constitutively activated. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines which act as docking sites for a number of SH2-containing molecules. These include Grb2 and p85 which couple the receptor, respectively, with Ras and PI 3-kinase. We previously showed that a Met mutant designed to obtain preferential coupling with Grb2 (Met2xGrb2) is permissive for motility, increases transformation, but - surprisingly - is impaired in causing invasion and metastasis. In this work we used Met mutants optimized for binding either p85 alone (Met2xPI3K) or p85 and Grb2 (MetPI3K/Grb2) to evaluate the relative importance of Ras and PI 3-kinase as downstream effectors of Met. Met2xPI3K was competent in eliciting motility, but not transformation, invasion, or metastasis. Conversely, MetP13K/Grb2 induced motility, transformation, invasion and metastasis as efficiently as wild type Met. Furthermore, the expression of constitutively active PI 3-kinase in cells transformed by the Met2xGrb2 mutant, fully rescued their ability to invade and metastasize. These data point to a central role for PI 3-kinase in Met-mediated invasiveness, and indicate that simultaneous activation of Ras and PI 3-kinase is required to unleash the Met metastatic potential.  (+info)

TIF1gamma, a novel member of the transcriptional intermediary factor 1 family. (18/190738)

We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma. Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences. In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression.  (+info)

Molecular phylogeny of the ETS gene family. (19/190738)

We have constructed a molecular phylogeny of the ETS gene family. By distance and parsimony analysis of the ETS conserved domains we show that the family containing so far 29 different genes in vertebrates can be divided into 13 groups of genes namely ETS, ER71, GABP, PEA3, ERG, ERF, ELK, DETS4, ELF, ESE, TEL, YAN, SPI. Since the three dimensional structure of the ETS domain has revealed a similarity with the winged-helix-turn-helix proteins, we used two of them (CAP and HSF) to root the tree. This allowed us to show that the family can be divided into five subfamilies: ETS, DETS4, ELF, TEL and SPI. The ETS subfamily comprises the ETS, ER71, GABP, PEA3, ERG, ERF and the ELK groups which appear more related to each other than to any other ETS family members. The fact that some members of these subfamilies were identified in early metazoans such as diploblasts and sponges suggests that the diversification of ETS family genes predates the diversification of metazoans. By the combined analysis of both the ETS and the PNT domains, which are conserved in some members of the family, we showed that the GABP group, and not the ERG group, is the one most closely related to the ETS group. We also observed that the speed of accumulation of mutations in the various genes of the family is highly variable. Noticeably, paralogous members of the ELK group exhibit strikingly different evolutionary speed suggesting that the evolutionary pressure they support is very different.  (+info)

ETO-2, a new member of the ETO-family of nuclear proteins. (20/190738)

The t(8;21) is associated with 12-15% of acute myelogenous leukemias of the M2 subtype. The translocation results in the fusion of two genes, AML1 (CBFA2) on chromosome 21 and ETO (MTG8) on chromosome 8. AML1 encodes a DNA binding factor; the ETO protein product is less well characterized, but is thought to be a transcription factor. Here we describe the isolation and characterization of ETO-2, a murine cDNA that encodes a new member of the ETO family of proteins. ETO-2 is 75% identical to murine ETO and shares very high sequence identities over four regions of the protein with ETO (domain I-III and zinc-finger). Northern analysis identifies ETO-2 transcripts in many of the murine tissues analysed and in the developing mouse embryo. ETO-2 is also expressed in myeloid and erythroid cell lines. We confirmed the nuclear localization of ETO-2 and demonstrated that domain III and the zinc-finger region are not required for nuclear localization. We further showed that a region within ETO, containing domain II, mediates dimerization among family members. This region is conserved in the oncoprotein AML-1/ETO. The recent identification of another ETO-like protein, myeloid translocation gene-related protein 1, together with the data presented here, demonstrates that at least three ETO proteins exist with the potential to form dimers in the cell nucleus.  (+info)

Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S. (21/190738)

The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The p41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the p41 fragment, a homologue of the thyroglobulin type-1 domains, has been determined at 2.0 A resolution in complex with cathepsin L. The structure of the p41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an alpha-helix-beta-strand arrangement, whereas the second subdomain has a predominantly beta-strand arrangement. The wedge shape and three-loop arrangement of the p41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the p41 fragment results in additional contacts with the top of the R-domain of the enzymes, which defines the specificity-determining S2 and S1' substrate-binding sites. This enables inhibitors based on the thyroglobulin type-1 domain fold, in contrast to the rather non-selective cystatins, to exhibit specificity for their target enzymes.  (+info)

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D. (22/190738)

The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.  (+info)

A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA. (23/190738)

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.  (+info)

Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection. (24/190738)

A new insect member of the STAT family of transcription factors (Ag-STAT) has been cloned from the human malaria vector Anopheles gambiae. The domain involved in DNA interaction and the SH2 domain are well conserved. Ag-STAT is most similar to Drosophila D-STAT and to vertebrate STATs 5 and 6, constituting a proposed ancient class A of the STAT family. The mRNA is expressed at all developmental stages, and the protein is present in hemocytes, pericardial cells, midgut, skeletal muscle and fat body cells. There is no evidence of transcriptional activation following bacterial challenge. However, bacterial challenge results in nuclear translocation of Ag-STAT protein in fat body cells and induction of DNA-binding activity that recognizes a STAT target site. In vitro treatment with pervanadate (vanadate and H2O2) translocates Ag-STAT to the nucleus in midgut epithelial cells. This is the first evidence of direct participation of the STAT pathway in immune responses in insects.  (+info)