The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell.

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.

ATF4 degradation relies on a phosphorylation-dependent interaction with the SCF(betaTrCP) ubiquitin ligase.

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein betaTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkappaBalpha and beta-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of betaTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of betaTrCP. The F-box-deleted betaTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF(betaTrCP) complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.

A novel Toxoplasma gondii calcium-dependent protein kinase.

Toxoplasma gondii is an obligate intracellular parasite that infects all types of cells in humans. A family of calcium-dependent protein kinases (CDPKs), previously identified as important in the development of plants and protists, was recently shown to play a role in the infectivity of apicomplexans, and in motility and host cell invasion in particular. We report here the isolation of a new calcium-dependent protein kinase gene from the human toxoplasmosis parasite, Toxoplasma gondii. The gene consists of 12 exons. The encoded protein, TgCDPK4, consists of the four characteristic domains of members of the CDPK family and is most similar to PfCDPK2 from Plasmodium falciparum. We measured TgCDPK4 activity, induced by calcium influx, using a kinase assay. A calcium chelator (EGTA) inhibited this activity. These findings provide evidence of signal transduction involving members of the CDPK family in T. gondii.

The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling.

BACKGROUND: Human immunodeficiency virus (HIV) Nef protein accelerates virulent progression of acquired immunodeficiency syndrome (AIDS) by its interaction with specific cellular proteins involved in signal transduction and host cell activation. Nef has been shown to bind specifically to a subset of the Src family of kinases. The structures of free Nef and Nef bound to Src homology region 3 (SH3) domain are important for the elucidation of how the affinity and specificity for the Src kinase family SH3 domains are achieved, and also for the development of potential drugs and vaccines against AIDS. RESULTS: We have determined the crystal structures of the conserved core of HIV-1 Nef protein alone and in complex with the wild-type SH3 domain of the p59fyn protein tyrosine kinase (Fyn), at 3.0 A resolution. Comparison of the bound and unbound Nef structures revealed that a proline-rich motif (Pro-x-x-Pro), which is implicated in SH3 binding, is partially disordered in the absence of the binding partner; this motif only fully adopts a left-handed polyproline type II helix conformation upon complex formation with the Fyn SH3 domain. In addition, the structures show how an arginine residue (Arg77) of Nef interacts with Asp 100 of the so-called RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the loop to adapt to complement the Nef surface. The Arg96 residue of the Fyn SH3 domain is specifically accommodated in the same hydrophobic pocket of Nef as the isoleucine residue of a previously described Fyn SH3 (Arg96-->lle) mutant that binds to Nef with higher affinity than the wild type. CONCLUSIONS: The three-dimensional structures support evidence that the Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor signaling. The structures of bound and unbound Nef reveal that the multivalency of SH3 binding may be achieved by a ligand induced flexibility in the RT loop. The structures suggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.

Alternative splicing in the human lck gene leads to the deletion of exon 1' and results in a new type II lck transcript.

In this study we describe a new lck mRNA transcribed from the type II promoter, resulting from alternative splicing, in the deletion of exon 1' which comprises the AUG initiation codon. In this type IIB lck transcript, as a consequence of exon 1' deletion, a new AUG codon in exon 1, located 35 nt upstream from the regular initiation codon in exon 1', and normally out of frame, is now in frame with exon 2 and the following lck coding sequence. In this type IIB lck transcript, 10 residues encoded by exon 1 from the new AUG codon replace the first 35 residues encoded by exon 1'. Strikingly, in this new N-terminal amino acid sequence, a glycine residue, needed for myristylation and anchorage in the plasma membrane, is present at position 2. This alternative splicing has been observed in T-cell lines, normal mature T cells and in peripheral blood lymphocytes from different leukemic patients. Different ratios of regular type IIA lck mRNA (with exon 1') to spliced type IIB lck mRNA (without exon 1') have been observed depending on the patient.

A single base mutation in the 5' splice site of intron 7 of the lck gene is responsible for the deletion of exon 7 in lck mRNA of the JCaM1 cell line.

The failure of signal transduction in the JCaM1 cell line was associated with the presence of an abnormal lck mRNA deleted of the exon 7 encoding for an inactive p56lck kinase. Our study of the lck mRNA from various T cell lines and from peripheral blood lymphocytes of healthy donors has revealed the presence of both complete and exon 7-deleted lck transcripts. Thus the exon 7-deleted lck transcript initially described in the JCaM1 mutant cell line, arises from an alternative splicing event occurring in each cells expressing the lck gene. Genomic DNA sequencing of the lck exons 6-8 portion from both the mutant JCaM1 and its parental Jurkat cell lines revealed as the only difference, the presence of a A to G mutation within the 5' splice site of intron 7 in the JCaM1 cell line DNA. To demonstrate the role of this point mutation in the lck pre-mRNA maturation, COS cells were transfected by lck minigenes from the Jurkat and JCaM1 cell lines. In COS cells transfected with minigene from the Jurkat cell line both lck transcripts (with and without exon 7) were observed whereas only the exon 7-spliced lck transcript was observed in COS cells transfected with minigene from the JCaM1 cell line. Thus the mutation is per se responsible for the deletion of exon 7 and the absence of complete lck mRNA in the JCaM1 cell line. Presence of a restriction site (HphI) in the 5' splice site of lck intron 7 from Jurkat DNA allowed to confirm the presence of the mutation on both alleles in the JCaM1 cell line.

Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s).

The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family. This phosphorylation site has a negative regulatory function. Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity. Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein. We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s). GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro. The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase. This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro. These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins.

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.

Cytoplasmic leucyl-tRNA synthetase of Neurospora crassa is not specified by the leu-5 locus.

We generated a lambda gt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, lambda NCLRSC1 and lambda NCLRSC2, were obtained which have inserts of approximately 2 kbp and approximately 1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that lambda NCLRSC1 and lambda NCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by lambda NCLRSC1 or lambda NCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an approximately 115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is approximately 3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located.

Selective increase of alternatively spliced Lck transcripts from the proximal promotor in hematopoietic malignancies.

Screening by Northern blot for lck expression in 51 patients with diverse hematologic diseases has shown, for four of them, a 3 to 15-fold increase in the level of lck mRNA when compared with expression in healthy donors. These patients suffered from diverse malignancies: one Burkitt lymphoma, one T-cell lymphoma and two non-Hodgkin B-cell lymphoma. Specific analysis of the different lck transcripts in these patients by polymerase chain reaction and their relative quantitation demonstrate a significant increase of only the type IB and the type IC lck transcripts arising from the proximal promotor. Our study shows: (a) a high lck expression may occur in diverse hematologic diseases, (b) whatever the type of malignancy, this high expression results in a specific increase of the spliced transcripts arising only from the proximal promotor, and (c) in these four patients without any rearrangement or amplification, the high lck expression probably results from the specific activation of the proximal promotor.

Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor.

The ex vivo approach to hepatic gene therapy involves several steps, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy.

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

Structure of the human lck gene: differences in genomic organisation within src-related genes affect only N-terminal exons.

Although cDNA sequences coding for several Rous sarcoma virus Src-related protein tyrosine kinases (PTKs) have been reported for several years, knowledge of the structure and organisation of genes of the src family is still limited. In this work, a detailed structure and organisation of the human lck gene is reported. A 17-kb genomic clone encoding human p56 Lck, a lymphocyte-specific PTK of the Src-related subfamily, has been isolated. The human lck gene is organized in 13 exons, one more than in the human cellular (c)-src gene. The twelve coding exons are located in this clone, whereas the putative 5'-noncoding exon is probably located very far upstream from the second exon. Splicing sites for exons 4 to 12, which encode both conserved phospholipase-C-like and catalytic domains of the Src-like PTKs, arise exactly at the same position for the human lck, human c-src and c-fgr genes. The only differences concern the splice sites of exons 1' and 2, which encode the unique N-terminal domain of human Lck. These results give further evidence that the different PTKs of the Src-like family have probably evolved through the mechanism of exon shuffling.

Use of a constrain phage displayed-peptide library for the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7).

It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.

Characterization and regulation of the angiotensin II type-1 receptor (binding and mRNA) in human adrenal fasciculata-reticularis cells.

The classical concept of human adrenal physiology indicates that only glomerulosa cells are the target of A-II. Herein, we demonstrated that cultured human adrenal fasciculata-reticularis cells were also responsive to this hormone. Indeed, these cells contained high affinity (Kd = 0.9-1.1 nM) and low capacity (8,000-13,000 sites/cell) A-II receptors, and more than 95% of them were of the type-1. These AT1 receptors are functional since A-II was able to increase cortisol production after 48 h of treatment. These effects were inhibited by losartan, an AT1 antagonist, but not by CGP42112A, an AT2 antagonist. The expression of the type-1 A-II receptor mRNA was detected in the whole adrenal in both adult and fetus, and in cultured human adrenal fasciculata-reticularis cells. In these cells A-II negatively regulated AT1 receptor mRNA, and this effect was also mediated through the AT1 receptor subtype.

Spleen protein tyrosine kinases TPK-IIB and CSK display different immunoreactivity and opposite specificities toward c-src-derived peptides.

Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48-64 and 353-369 sequences of CSK, a protein tyrosine kinase implicated in the down-regulation of src-related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK-related 49 kDa protein tyrosine kinase present in spleen but they do not cross-react with purified TPK-IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK-IIB once resolved from each other by heparin-Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp60c-src, namely Tyr-416 and Tyr-527. These data support the view that TPK-IIB and CSK may exert opposite effects on the activity of src-related protein tyrosine kinases.

Ca++ binding properties of human prothrombin.

The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin.

Identification of a mouse cDNA fragment whose expressed polypeptide reacts with anti-recA antibodies.

We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein. Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies. The cDNA fragment is 601 nucleotide long and was called KIN17(601). It contains an open reading frame coding for a 200 amino acid polypeptide. In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein. Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins.

An anti-HIV-1 gag protein rat monoclonal antibody library.

A set of 18 rat monoclonal antibodies (MAbs) directed against human immunodeficiency virus type 1 (HIV-1) gag proteins was derived from 4 independent fusion protocols. The epitopes recognized were delineated using a random fragment expression library representing the whole HIV-1IIIB genome. This panel of rat MAbs was used to analyze the antigenicities of the HIV-1 CAp24 major core protein and the HIV-1 NCp7 nucleocapsid protein. As a result, a limited set of antigenic domains as defined, 3 on CAp24 between amino acids (aa) 195 and 268, 323 and 329, 329 and 352, and one on NCp7 (aa 382-392). Only 4 mouse anti-CAp24 MAbs appeared to recognize the COOH-terminal domain (aa 329-352) defined by the majority of our MAbs. The rat anti-CAp24 (Q1B10) and the rat anti-NCp7 (I5B11) MAbs described here, defined two newly described epitopes, aa 323-329 and aa 382-392.

Isolation of preliminary characterization of a vitamin K dependent peptide from human prothrombin.

A peptide containing the vitamin K dependent Ca2+ binding region of human prothrombin has been isolated by adsorption of the tryptic digest of the native protein on barium citrate. Its aminoacid composition has been determined. The N- and C-terminal residues have been characterized. The obtained results are showing some slight differences with the corresponding bovine peptide. Since they were not strictly identical for two different preparations, the eventuality of a molecular heterogeneity is discussed.