Identification of alternatively spliced Dab1 and Fyn isoforms in pig

Abstract

BackgroundDisabled-1 (Dab1) is an adaptor protein that is essential for the intracellular transduction of Reelin signaling, which regulates the migration and differentiation of postmitotic neurons during brain development in vertebrates. Dab1 function depends on its tyrosine phosphorylation by Src family kinases, especially Fyn.ResultsWe have isolated alternatively spliced forms of porcine Dab1 from brain (sDab1) and liver (sDab1-Li) and Fyn from brain (sFyn-B) and spleen (sFyn-T). Radiation hybrid mapping localized porcine Dab1 (sDab1) and Fyn (sFyn) to chromosomes 6q31-35 and 1p13, respectively. Real-time quantitative RT-PCR (qRT-PCR) demonstrated that different isoforms of Dab1 and Fyn have tissue-specific expression patterns, and sDab1 and sFyn-B display similar temporal expression characteristics in the developing porcine cerebral cortex and cerebellum. Both sDab1 isoforms function as nucleocytoplasmic shuttling proteins. It was further shown that sFyn phosphorylates sDab1 at tyrosyl residues (Tyr) 185, 198/200 and 232, whereas sDab1-Li was phosphorylated at Tyr 185 and Tyr 197 (corresponding to Y232 in sDab1) in vitro.ConclusionsAlternative splicing generates natural sDab1-Li that only carries Y185 and Y197 (corresponding to Y232 in sDab1) sites, which can be phosphorylated by Fyn in vitro. sDab1-Li is an isoform that is highly expressed in peripheral organs. Both isoforms are suggested to be nucleocytoplasmic shuttling proteins. Our results imply that the short splice form sDab1-Li might regulate cellular responses to different cell signals by acting as a dominant negative form against the full length sDab1 variant and that both isoforms might serve different signaling functions in different tissues.

Splice-Mediated Motif Switching Regulates Disabled-1 Phosphorylation and SH2 Domain Interactions

“…We did not detect either Dab1 217 or Dab1 271 in our study; however, it should be noted that both of these isoforms introduce in-frame premature termination codons (PTC), which may promote RNA degradation through nonsense-mediated mRNA decay (NMD) (5). Dab1 variant 5 contains exons 9b/c (3,20,46), whose homologues have also been detected in chicken, lizard, and pig (3,29,31). Unlike the inclusion of exons 9b/9c in mouse variant 5, 9b/9c inclusion in chicken and pig is accompanied by concurrent exclusion of exons 7 and 8, producing an isoform homologous to the variant 6 identified in the present study.…”

“…The genomic organization of Dab1 is unusually complex and highly conserved in different species (3,10,31 (3,20,46). All three Dab1 isoforms retain the intact PI/PTB domain at the N terminus but differ in the middle tyrosine-enriched region or in the C terminus.…”

“…When alternative splicing occurs at these exons, it generates isoforms with modified tyrosine phosphorylation sites. For example, alternative splicing of exons 7 and 8, which removes the Y 198 Q and Y 220 Q residues and converts the Y 185 QXI motif into a Y 185 QVP motif, has been documented in chick retina, human retina and pig liver (28)(29)31). Skipping of exons 8 and 9, which removes both Y 220 QVP and Y 232 QVP, has been observed in zebrafish brain (10).…”

Reelin-Disabled-1 signaling in neuronal migration: splicing takes the stage

“…Notably, splicing of exons 9b/9c and exons 7/8 is often mutually exclusive [69,76], suggesting coordinated regulation of the exclusion and inclusion of these exons during development. It would be of interest to investigate whether Nova also promotes exons 7/8 inclusion along with exons 9b/9c exclusion, and whether isoforms that exclude exons 7/8 are increased in Nova2 −/− mice along with isoforms that include exons 9b/9c.…”

Fyn Regulates Adipogenesis by Promoting PIKE-A/STAT5a Interaction

“…Nevertheless, scattered pieces of information also show that Fyn may play a role in adipogenesis in addition to regulating energy expenditure. A recent study performed in pig indicates that a substantial level of Fyn mRNA can be detected in WAT (14). Using a comparative functional genomics approach, it has also been reported that the adipose Fyn expression is increased in obese rats (15).…”