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PubMed1999

Persistent expression of developmental myosin heavy chain isoforms in the tapered ends of adult pigeon pectoralis muscle fibres.

We have shown previously that in addition to the adult myosin heavy chain (MyHC) isoform present throughout the length of each fast-twitch glycolytic muscle fibre within the pectoralis of the mature chicken, the neonatal isoform is retained in the tapered ends of these fibres. This work, however, has been the only published report of this phenomenon. Here, we tested the hypothesis that similar to the chicken, the ends of mature pigeon pectoralis muscle fibres contain developmental MyHC isoform(s). A histological stain was used to visualize endomysium to assist in the analysis of transverse sections of pectoralis muscle from four mature pigeons. Immunocytochemical techniques were used to localize MyHC isoform(s) characteristic of pigeon pectoralis development. We show that within mature pigeon pectoralis, the ends of both fast-twitch glycolytic and fast-twitch oxidative-glycolytic fibre types express MyHC isoform(s) characteristic of their earlier development. Thus, we extend our findings on chicken to another species and an additional muscle fibre type. Retention of developmental MyHC isoform(s) within the tapered ends of mature muscle fibres may be more widespread than is currently appreciated.

Animals↗
PubMed2001

Expression of Ca2+/calmodulin-dependent protein kinase II delta-subunit isoforms in rats with hypertensive cardiac hypertrophy.

Myocardial hypertrophy is characterized by abnormal intracellular Ca2+ handling and decreased contractile performance. Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates numerous Ca2+ handling proteins and thus can regulate intracellular Ca2+ homeostasis directly. We therefore investigated whether differential expression of CaMKII isoforms occurs with cardiac hypertrophy which might promote an abnormal intracellular Ca2+ homeostasis. We further investigated the potential influence of angiotensin (Ang) II on CaMKII expression levels. Hearts from adult Spontaneously Hypertensive Rats (SHR) and hearts from two transgenic rat models with Ang II-dependent hypertension were studied. The expression of the cardiac CaMKII isoforms delta2, delta3, delta4 and delta9 was determined by RT-PCR and immunoblot methods. Rats transgenic for the mouse Ren-2 gene (mrTGR), SHR and controls were studied at the age of 6 months and rats transgenic for the human renin-angiotensin system (hrTGR) from postnatal day 1 to week 8. SHR and mrTGR had an increased heart/body weight ratio (26 and 25%) compared with controls (p < 0.05). SHR hearts showed significantly increased mRNA levels of delta4 and delta9 (p < 0.05) with no change for delta2 and delta3. mrTGR hearts had a significantly increased delta4 and a significantly decreased delta3 transcript level (p < 0.05) with no change for delta2 and delta9. hrTGR hearts developed severe hypertrophy (42%) after postnatal day 14. The neonatal delta2, delta3 and delta4 isoform expression levels were higher (30-100%) compared with SD controls. The levels decreased with increasing age and equalized to controls at week 8, except for delta4 which started to increase after week 4 (p < 0.05). CaMKIIdelta protein levels of all cardiac hypertrophy models were increased in sarcoplasmic reticulum preparations (50-120%) compared with controls (p < 0.05) while the cytosolic levels remained unchanged. Thus, CaMKIIdelta isoforms are differentially expressed in cardiac hypertrophy. The fetal delta4 isoform was constantly expressed. CaMKIIdelta adopts the fetal phenotype independent of the type of hypertrophic stimulus. The observed alterations of CaMKIIdelta isoform patterns may affect intracellular Ca2+ homeostasis and thus contribute to the abnormal contractile phenotype of cardiac hypertrophy.

Age Factors↗
PubMed2001

Myosin and SERCA isoform expression in denervated slow-twitch muscle of euthyroid and hyperthyroid rabbits.

Changes in the isoform patterns of myosin heavy chains (MHC) and Ca2+-ATPase (SERCA) were studied in long-term (72 days) denervated slow-twitch soleus muscles of euthyroid and hyperthyroid rabbits. MHC isoforms were separated electrophoretically. SERCA isoforms were assessed by electrophoretic separation of the trypsin-digested and phosphorylated fragments. Denervation led to pronounced slow-to-fast transitions with increases in the fast MHC and fast SERCA1a isoforms. Hyperthyroidism had no significant effect on the denervation-induced changes in SERCA isoforms, but led to slight increases in the relative concentrations of the fast MHC isoforms. A high correlation existed between the relative concentrations of slow myosin and SERCA2a in both innervated euthyroid and hyperthyroid soleus muscles. This correlation, however, seems to be less tight in denervated soleus muscles.

Animals↗
PubMed2000

Genomic characterization of CD84 reveals the existence of five isoforms differing in their cytoplasmic domains.

CD84, a new member of the immunoglobulin superfamily, shows high homology with several molecules belonging to the CD2 family of differentiation antigens. By screening a peripheral blood leukocyte cDNA library four CD84 isoforms were obtained differing in their 3' sequence. A reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that these isoforms were normally found on leukocytes and a new isoform was identified. To establish the nature of the five isoforms obtained (CD84a, CD84b, CD84c, CD84d and CD84e) the genomic structure of the CD84 gene was determined. Our results show that it is composed of at least eight exons, with an exon coding for the 5' UTR and the leader peptide, two exons coding for each of the two immunoglobulin-like domains, an exon encoding the transmembrane portion and four exons coding for the cytoplasmic domains. The isoforms are generated by several mechanisms: alternative use of exons, reading frame shift, use of a cryptic splice site or absence of splicing. The differential expression of several potentially phosphorylatable residues on the different isoforms could be a way to regulate its possible activity in signal transduction.

Amino Acid Sequence↗
PubMed2005

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms.

The repeat region of DC-SIGNR (CD209L) is polymorphic on the genomic level, and, in a separate study, we observed a correlation between the DC-SIGNR genotype and HIV-1 susceptibility during sexual contact. However, previous investigations using immunohistochemistry failed to detect membrane-bound DC-SIGNR on cells in the genital and rectal mucosa. We therefore explored the presence of DC-SIGNR in these compartments with a more sensitive limiting dilution RT-PCR, which also allowed for quantification of alternatively spliced mRNA isoforms. DC-SIGN (CD209) and DC-SIGNR mRNA transcript isoforms were found in all 12 vaginal and two rectal biopsies obtained from 14 healthy individuals. For DC-SIGNR, we detected significantly more isoform than full-length transcripts (mean copy numbers/mug RNA: 602 vs 26; P=0.0009). Four mucosal samples lacked full-length DC-SIGNR transcripts entirely. Cloning and sequencing of DC-SIGNR mRNA in three additional individuals revealed a diverse repertoire of DC-SIGNR isoforms, many of which encoded for proteins predicted to be soluble and secreted. Indeed, in one vaginal sample, we detected only soluble isoforms. In conjunction with our prior observation that the DC-SIGNR genotype has an effect on HIV-1 transmission in vivo, these findings emphasize that DC-SIGNR, in addition to DC-SIGN, should be considered as a cofactor in sexual HIV-1 transmission. Soluble isoforms, in particular, may modulate the efficiency of viral transmission and dissemination.

Amino Acid Sequence↗
PubMed2006

Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium.

Previous studies have identified mRNA three isoforms encoding interleukin-15 (IL-15) that are produced through differential splicing and encode for the same mature IL-15 protein with two different signal peptides. Our analysis of mouse intestinal epithelial cells revealed two new IL-15 mRNA isoforms generated by different alternative splicing events. In one form (IL-15DeltaE6), exon 6 is absent, and in the second form the first 48 nt of exon 7 are absent (IL-15DeltaE7) through usage of an alternative 5' splicing site within exon 7. These mRNA isoforms encoded in-frame IL-15 protein variants lacking either 15aa (IL-15DeltaE6) or 16aa (IL-15DeltaE7) both utilizing the normal long signal peptide. Significant structural changes were predicted for these new IL-15 isoforms. RNAse protection assays revealed the highest expression of isoform mRNA in the intestinal epithelium and functional analysis of recombinant IL-15 isoform proteins suggested possible regulatory functions.

Alternative Splicing↗
PubMed1999

Distinct expression patterns and transforming properties of multiple isoforms of Ost, an exchange factor for RhoA and Cdc42.

A search for transforming genes expressed in brain led to the identification of a novel isoform of Ost, an exchange factor for RhoA and Cdc42. In addition to the Dbl-homology (DH) and pleckstrin-homology (PH) domains identified in the original Ost, this isoform contained a SH3 domain and a novel HIV-Tat related (TR) domain. The presence or absence of these domains in Ost defined multiple isoforms of the protein. RT - PCR and in situ hybridization analysis revealed that these isoforms were generated by tissue-specific and developmentally restricted alternative splicing events. Whereas deletion of the N-terminus activated the transforming properties of Ost, the presence of the SH3 domain reduced the transforming activity of the protein. This inhibition was relieved by the presence of a TR domain, which contained a potential SH3 ligand sequence. The transforming activity of all Ost isoforms was inhibited by dominant negative forms of the Rho family proteins. Expression of Ost isoforms potently induced the formation of actin stress fibers and filopodia as well as JNK activity and AP1- and SRF-regulated transcriptional pathways. Ost transfectants also displayed elevated levels of cyclins A and D1, suggesting that the de-regulation of these cyclins is linked to Ost-mediated transformation.

3T3 Cells↗
PubMed2002

Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-1-transfected C33A human cervical carcinoma cell line.

BAG-1 protein can be expressed as four isoforms of 50, 46, 33 and 29 kDa with different subcellular localizations, which may have different functions in anti-apoptosis, but the exact mechanism remains unclear. We constructed BAG-1 full length and deletion mutated plasmids in a pCR3.1 vector and established stable transfections of BAG-1 isoforms in low BAG-1 expressing C33A cells. Treatment of the transfected cells with cisplatin, staurosporine, paclitaxel and doxorubicine showed that BAG-1 p50, p46 and p33 isoforms enhanced the resistance to apoptosis. BAG-1 p50, p46 and p33 exhibited different degrees of apoptosis inhibition in the transfected cells and BAG-1 p46 isoform had the most pronounced effect on anti-apoptosis. BAG-1 p29 failed to protect the transfected cells from apoptosis. Resistance to apoptosis by BAG-1 isoforms was correlated with decreased caspase-3 activation. We also detected the expression of Bax, Bak, p53, Bcl-2, Bcl-X(L), AIF and MRP1 by Western blots. Bcl-2 protein expression was significantly increased in p50, p46 and p33 transfected cells, while the expression of Bax, Bak, p53, Bcl-X(L) and MRP1 was essentially unchanged. These in vitro results suggest that distinct isoforms of BAG-1 have different anti-apoptotic functions and their functions may be correlated to increased Bcl-2 expression.

Apoptosis↗
PubMed2006

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(-)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(-)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Apoptosis↗
PubMed2002

Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors.

We identified a thyroid hormone [3,5,3'-tri-iodothyronine (T(3))]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, alpha, beta1 and beta2, in human brain by 5'- and 3'-RACE (rapid amplification of cDNA ends). The alpha transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that beta1 and beta2 encode an identical protein product, beta, which differs from alpha in its N-terminus. Since alpha and beta contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the alpha transcript is expressed exclusively in the brain, whereas beta transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both alpha and beta transcripts, raising the question of which transcript is up-regulated by T(3). It was revealed that T(3) markedly induced the expression of alpha isoform but not of beta. This T(3)-mediated increase in the alpha isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.

Alternative Splicing↗
PubMed2003

Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration.

SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY(246)AQL and VVY(263)ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34(+)CD38(+) and early clonogenic progenitors, and at lower levels on CD34(+)CD38(-) cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133(+) precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Delta 46-110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility.

Amino Acid Motifs↗
PubMed2002

Specificity of 14-3-3 isoform dimer interactions and phosphorylation.

Proteins that interact with 14-3-3 isoforms are involved in regulation of the cell cycle, intracellular trafficking/targeting, signal transduction, cytoskeletal structure and transcription. Recent novel roles for 14-3-3 isoforms include nuclear trafficking the direct interaction with cruciform DNA and with a number of receptors, small G-proteins and their regulators. Recent findings also show that the mechanism of interaction is also more complex than the initial finding of the novel phosphoserine/threonine motif. Non-phosphorylated binding motifs that can also be of high affinity may show a more isoform-dependent interaction and binding of a protein through two distinct binding motifs to a dimeric 14-3-3 may also be essential for full interaction. Phosphorylation of specific 14-3-3 isoforms can also regulate interactions. In many cases, they show a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of dimer formation may influence which of the 14-3-3-interacting proteins could be brought together. Mammalian and yeast 14-3-3 isoforms show a preference for dimerization with specific partners in vivo.

14-3-3 Proteins↗
PubMed1998

Characterization of serum SHBG isoforms in prepubertal and pubertal girls.

OBJECTIVE: SHBG is a circulating glycoprotein that binds dihydrotestosterone, testosterone and oestradiol with high affinity and low capacity. In girls, serum concentrations of SHBG gradually decrease with age due to a true fall in concentration and not to a change in the binding characteristics. The aim of our study was to determine the pattern of serum SHBG isoforms in normal girls in early childhood (ECh), late childhood (LCh) and puberty (P). SUBJECTS: Fifteen normal girls were studied. They were divided into three groups according to their age: ECh: 3.7 +/- 0.9 years (mean +/- SD, n = 5); LCh: 6.4 +/- 0.5 years (n = 5); and P: 13.4 +/- 1.5 years (n = 5). METHODS AND MEASUREMENTS: Preparative isoelectric focusing was used to isolate SHBG isoforms according to their isoelectric point (pI). Three groups of isoforms were isolated: SI: pI 5.2-5.4; SII: pI 5.4-5.6 and SIII: pI 5.6-5.8. Steroid levels in serum were determined by RIA. RESULTS: The relative distribution of SHBG isoforms (% of the total SHBG recovered, mean +/- SD) in the three groups of girls studied was: ECh: SI: 25.8 +/- 9.9, SII: 53 +/- 10.5 and SIII: 21.2 +/- 1.6; LCh: SI: 8.8 +/- 3.1, SII: 58.8 +/- 12.2 and SIII: 31.8 +/- 8.6; P: SI: non-detectable; SII: 51.6 +/- 12.6 and SIII: 48.4 +/- 12.6. CONCLUSION: These results indicate that serum SHBG is more heterogeneous before puberty. A considerable proportion of acidic isoforms are present early in life; they decrease during the prepubertal period and disappear when sexual development is completed. After puberty the glycoprotein is more homogeneous and an important proportion of more basic isoforms is present. At puberty serum SHBG not only falls in concentration but also has an altered sialic acid content which modulates its circulating half-life.

Adolescent↗
PubMed2002

Biglycan isoforms with differences in polysaccharide substitution and core protein in human lung fibroblasts.

Biglycan is widely distributed in the extracellular matrix and is a member of the small proteoglycan family characterized by a core protein with leucine-rich repeat motifs. We show in this paper for the first time that biglycan from human lung fibroblasts can be expressed as different isoforms. These isoforms can be separated from the predominant form of biglycan by hydrophobic interaction chromatography, where the more hydrophobic isoforms are retarded. The newly found isoforms of biglycan have a smaller core protein substituted with smaller glycosaminoglycan chains, migrating on SDS/PAGE at between 110 and 200 kDa. These molecules were identified as biglycan using MALDI-TOF MS. Identification of C-terminal peptides together with glycosylation of the N-terminal glycosaminoglycan sites excludes the possibility of terminal proteolytic cleavage. The biglycan isoforms are N-glycosylated, which demonstrates that a lack in N-glycosylation is not the reason for a smaller core. Two components revealed by RT-PCR indicate alternative splicing, which could be located in regions of the protein that have not been identified, with the exclusion of sites of glycosylations. Analyses of glycosaminoglycan chain length of the isoforms show that besides the normally occurring glycosaminoglycan chains, there is a mixture of shorter glycosaminoglycan chains. Structural analysis shows that these glycosaminoglycan chains contain a lower proportion of iduronic acid (61%) relative to glucuronic acid when compared to the glycosaminoglycan chain of the predominant form of biglycan (71%). We can anticipate that variation in structure of biglycan can cause changes in the connective tissue formation depending on its ability to bind matrix molecules, as well as cytokines.

Alternative Splicing↗
PubMed1999

Striking multiplicity of eIF4E-BP1 phosphorylated isoforms identified by 2D gel electrophoresis regulation by heat shock.

Eukaryotic initiation factor eIF4E-binding protein 1 (eIF4E-BP1), or PHAS-I, is multiply phosphorylated by insulin-stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E-BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E-BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8-10 prominent eIF4E-BP1 isoforms were detected. Following heat shock, a rapid, temperature-dependent dephosphorylation of eIF4E-BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E-BP1 parallels restoration of protein synthesis. However, eIF4E-BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E-BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E-BP1. The amount of eIF4E-BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E-BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E-BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E-BP1)-specific antibodies. These results identify a complex set of eIF4E-BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

Amino Acid Motifs↗
PubMed2001

Characterization of a new brain-specific isoform of the EWS oncoprotein.

EWS and related TAFII68 and TLS/FUS genes are fused with different genes encoding transcription factors in various human cancers. The products of these genes have the ability to bind RNA and have been shown to be part of splicing and transcription complexes. We show that the EWS, TAFII68 and TLS/FUS proteins are expressed to various levels in all adult murine tissues. We characterize a new isoform of EWS that is specifically expressed in the central nervous system, in both mice and humans. It is shown to be related to a splice variant which includes a new 18-bp exon, termed 4', between exon 4 and 5. The detection of this isoform in spontaneously differentiating SH-SY5Y neuroblastoma cells and in nerve growth factor-induced PC12 cells further links this isoform to neural differentiation. RT-PCR experiments indicate that the level of expression of the brain-specific EWS isoform is stable during brain development whereas that of the ubiquitous EWS isoform decreases during this period. The two isoforms show a parallel decrease in expression after birth. The 4' exon is not detected in tumour-specific EWS fusion transcripts, suggesting that its presence may impair their oncogenic properties. Interestingly, sequences of the 4' exon and flanking regions show remarkable similarities to that of the neural-specific c-src exon, suggesting common mechanisms for the alternative splicing of these exons. The phylogenetic conservation and relationship to neural differentiation strongly suggests an important functional role for this exon.

Amino Acid Sequence↗
PubMed2002

Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis.

BACKGROUND & AIMS: Little is known of the signaling events implicated in the induction of human enterocytic anoikis. In the present study, we analyzed the role of the stress-activated protein kinase p38 in this process. METHODS: Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of a dominant negative form) or beta1 integrins (antibody blocking), or by maintaining cells in suspension. Expression/activation parameters of p38 (isoforms alpha, beta, gamma, delta) and of the Fak/phosphatidylinositol-3-kinase (PI3-K)/Akt anoikis-suppressing pathways were analyzed. Kinase activities of p38 isoforms also were blocked by pharmacologic inhibitors or by overexpression of dominant-negative forms. RESULTS: (1) p38 activation is sustained transiently after induction of anoikis in both undifferentiated and differentiated enterocytes; (2) such sustenance of p38 activation is associated with a down-regulation of the Fak/PI3-K/Akt pathway; (3) distinct profiles of p38 isoform expression are exhibited by undifferentiated (alpha, beta, gamma) and differentiated (alpha, gamma, delta) enterocytes; (4) none of the 4 known p38 isoforms was found to promote cell survival in either differentiation state; and (5) only p38beta and p38delta are required specifically for anoikis in undifferentiated and differentiated cells, respectively. CONCLUSIONS: Distinct p38 isoforms play a major role in the induction of enterocytic anoikis and the regulation of such selective p38 isoform-mediated anoikis is linked with the state of cell differentiation. These data provide novel insights into the synchronized regulation of cell survival/death required in the epithelial renewal process along the human intestinal crypt-villus axis.

Anoikis↗