Truncated BRCA2 is cytoplasmic: Implications for cancer-linked mutations

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway

STAT1 is an essential transcription factor for macrophage activation by IFN-γ and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-α occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-γ-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-γ-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-α caused activation of p38 MAPK whereas IFN-γ did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-α production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKα and β but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-γ-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): Characterization of connective tissue growth factor as a member of the IGFBP superfamily

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40–60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an “IGFBP motif” (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1–6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

Homeobox gene Prx3 expression in rodent brain and extraneural tissues

Different cDNA clones encoding a rat homeobox gene and the mouse homologue OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains). Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the “aristaless domain.” Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum revealed marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also expressed the Prx3 gene. In the adult rat brain, Prx3 gene expression was restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation.

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1–56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Australian lungfish neurohypophysial hormone genes encode vasotocin and [Phe2]mesotocin precursors homologous to tetrapod-type precursors

In view of the well-established role of neurohypophysial hormones in osmoregulation of terrestrial vertebrates, lungfishes are a key group for study of the molecular and functional evolution of the hypothalamo-neurohypophysial system. Here we report on the primary structure of the precursors encoding vasotocin (VT) and [Phe2]mesotocin ([Phe2]MT) of the Australian lungfish, Neoceratodus forsteri. Genomic sequence analysis and Northern blot analysis confirmed that [Phe2]MT is a native oxytocin family peptide in the Australian lungfish, although it has been reported that the lungfish neurohypophysis contains MT. The VT precursor consists of a signal peptide, VT, that is connected to a neurophysin by a Gly-Lys-Arg sequence, and a copeptin moiety that includes a Leu-rich core segment and a glycosylation site. In contrast, the [Phe2]MT precursor does not contain a copeptin moiety. These structural features of the lungfish precursors are consistent with those in tetrapods, but different from those in teleosts where both VT and isotocin precursors contain a copeptin-like moiety without a glycosylation site at the carboxyl terminals of their neurophysins. Comparison of the exon/intron organization also supports homology of the lungfish [Phe2]MT gene with tetrapod oxytocin/MT genes, rather than with teleost isotocin genes. Moreover, molecular phylogenetic analysis shows that neurohypophysial hormone genes of the lungfish are closely related to those of the toad. The present results along with previous morphological findings indicate that the hypothalamo-neurohypophysial system of the lungfish has evolved along the tetrapod lineage, whereas the teleosts form a separate lineage, both within the class Osteichthyes.

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-ψ[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1α and aggst1β) reported within are interrupted by introns. The gene aggst1α contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1α are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1β gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n = 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.

Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine

An isoform of the mammalian renal type II Na/Pi-cotransporter is described. Homology of this isoform to described mammalian and nonmammalian type II cotransporters is between 57 and 75%. Based on major diversities at the C terminus, the new isoform is designed as type IIb Na/Pi-cotransporter. Na/Pi-cotransport mediated by the type IIb cotransporter was studied in oocytes of Xenopus laevis. The results indicate that type IIb Na/Pi-cotransport is electrogenic and in contrast to the renal type II isoform of opposite pH dependence. Expression of type IIb mRNA was detected in various tissues, including small intestine. The type IIb protein was detected as a 108-kDa protein by Western blots using isolated small intestinal brush border membranes and by immunohistochemistry was localized at the luminal membrane of mouse enterocytes. Expression of the type IIb protein in the brush borders of enterocytes and transport characteristics suggest that the described type IIb Na/Pi-cotransporter represents a candidate for small intestinal apical Na/Pi-cotransport.

Modification of EWS/WT1 functional properties by phosphorylation

In many human cancers, tumor-specific chromosomal rearrangements are known to create chimeric products with the ability to transform cells. The EWS/WT1 protein is such a fusion product, resulting from a t(11;22) chromosomal translocation in desmoplastic small round cell tumors, where 265 aa from the EWS amino terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. Herein, we find that EWS/WT1 is phosphorylated in vivo on serine and tyrosine residues and that this affects DNA binding and homodimerization. We also show that EWS/WT1 can interact with, and is a substrate for, modification on tyrosine residues by c-Abl. Tyrosine phosphorylation of EWS/WT1 by c-Abl negatively regulates its DNA binding properties. These results indicate that the biological activity of EWS/WT1 is closely linked to its phosphorylation status.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.