Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers.     

Alternative splicing products of the gene for a human nuclear actin-related protein,hArpNbeta/Baf53, that encode a protein isoform,hArpNbetaS, in the cytoplasm

A human nuclear actin-related protein, hArpNbeta/ Baf53, is a component of chromatin remodeling and histone acetyltransferase complexes. We identified two alternative splicing products of the gene for hArpNbeta/ Baf53. They encoded a protein isoform, hArpNbetaS; and its fusion product with green fluorescent protein was to be found in the cytoplasm, not the nucleus. The isoforms may contribute to functional regulation of these complexes.     

Alternative splicing of delta-like 1 homolog (DLK1) in the pig and human

Delta-like homolog 1 (DLK1), a paternally imprinted gene with several alternative splicing isoforms, is an important regulator of fetal and postnatal development. We report the sequence of porcine DLK1 (pDLK1) and examine the expression and alternative splicing isoforms in the pig (Sus scrofa) and human. DLK1-A was the sole isoform identified in human tissues and has been shown to be present in mouse and cattle. Surprisingly, DLK1-A was undetected in various tissues from fetal and postnatal pigs. Instead, DLK1-C2 was the most abundant isoform while DLK1-B was expressed to a lesser extent. In fractionated adipose tissue, pDLK1 was most highly expressed in the stromal-vascular cell fraction. In addition, total pDLK1 was highly expressed in fetal adipose tissue but dramatically decreased postnatally. Our data suggests that expression of DLK1-B and -C2 isoforms is sufficient for normal pig development. Furthermore, human and pig samples showed no alterations in species-specific splicing, but expression levels decreased with age, suggesting that regulation of expression, not splicing, is the most likely mechanism controlling the biological function of DLK1.     

Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling

Alternative splicing of ASI residues (Ala³⁴⁸¹–Gln³⁴⁸⁵) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(?)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr³⁴⁷¹–Gly³⁵⁰⁰) in the splice variants. Depolarisation-dependent Ca²⁺ release was enhanced by >50% in myotubes expressing ASI(?)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca²⁺ content were unaltered, while ASI(?)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an α-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.     

Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

Phenotypic differentiation patterns of the human monocyte/macrophage system

Summary In the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.     

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.     

Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting

Plasma membrane Ca(2+)-ATPases (PMCAs) are involved in local Ca(2+) signaling and in the spatial control of Ca(2+) extrusion, but how different PMCA isoforms are targeted to specific membrane domains is unknown. In polarized MDCK epithelial cells, a green fluorescent protein-tagged PMCA4b construct was targeted to the basolateral membrane, whereas a green fluorescent protein-tagged PMCA2b construct was localized to both the apical and basolateral domain. The PDZ protein-binding COOH-terminal tail of PMCA2b was not responsible for its apical membrane localization, as a chimeric pump made of an NH(2)-terminal portion from PMCA4 and a COOH-terminal tail from PMCA2b was targeted to the basolateral domain. Deletion of the last six residues of the COOH terminus of either PMCA2b or PMCA4b did not alter their membrane targeting, suggesting that PDZ protein interactions are not essential for proper membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane targeting of PMCA2. Only the "w" form, which contains a 45-amino acid residue insertion, showed prominent apical membrane localization. By contrast, the x and z splice variants containing insertions of 14 and 0 residues, respectively, localized to the basolateral membrane. The w splice insert was the crucial determinant of apical PMCA2 localization, and this was independent of the splice configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of alternative splicing within the first cytosolic loop of PMCA2 and help explain the selective enrichment of specific PMCA2 isoforms in specialized membrane compartments such as stereocilia of auditory hair cells.     

Alternative splicing produces a constitutively active form of human SREBP-1

We identified a novel alternative splicing event that constitutively produces a truncated active form of human sterol regulatory element-binding protein 1 (SREBP-1). A cDNA of this splicing variant (named SREBP-1Δ) contains a translational stop codon-encoding exon sequence between exons 7 and 8. It produces SREBP-1aΔ (470 a.a.) and SREBP-1cΔ (446 a.a.) proteins that lack transmembrane and C-terminal regulatory sequences necessary for localization of SREBP-1 to the endoplasmic reticulum. A luciferase reporter assay showed that SREBP-1aΔ and SREBP-1cΔ transactivated lipogenic gene promoters to the same extent as that induced by N-terminal active fragments of SREBP-1a and SREBP-1c, respectively. SREBP-1Δ mRNA is expressed in human cell lines as well as adipose and liver tissues. Expression levels ranged from 5% to 16% of total SREBP-1 expression. The ratio of SREBP-1Δ expression to total SREBP-1 expression in HepG2 cells was not affected by either insulin or high glucose treatment.