Identification of alternatively spliced Dab1 and Fyn isoforms in pig

Abstract

Background: Disabled-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.

Results: We 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.

Conclusions: Alternative 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.

Figure 1

Organization of the porcine Dab1 and Fyn genes. A: Comparison of alternatively spliced isoforms of sDab1. The middle row shows genomic organization of the coding region of porcine Dab1 gene, which includes the relative positions of all the putative exons (black boxes) and introns (blocked lines). Note that the actual lengths of these exons may slightly vary since the 5'-UTR or 3'-UTR acquired in our study was not complete and the lengths of introns are not respected in this schematic figure. B: Changes of tyrosines after alternative splicing of sDab1. C: Comparison of alternatively spliced isoforms of sFyn. The middle row shows the genomic organization of the coding region of porcine Fyn gene, which includes the relative positions of all the putative exons (black boxes) and introns (blocked lines).

Figure 2

Expression of sDab1 and sFyn isoforms (A: sDab1, B: sDab1-Li, C: sFyn-B, D: sFyn-T) in various pig tissues. Total RNA was extracted from 12 different tissues (He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; Ad: adipose; Mu: muscle; St: stomach; In: intestine; Co: cerebral cortex; Ce: cerebellum; Te: testicle) and subjected to real-time RT-PCR. The expression of sDab1 and sFyn was calculated as a percentage of GAPDH mRNA level in parallel. Data are means ± SEM of four independent experiments.

Figure 3

Expression of sDab1 (A) and sFyn-B (B) in porcine cerebral cortex and cerebellum at different developmental stages. Quantitative real-time RT-PCR was performed on mRNAs prepared from cerebral cortex and cerebellum of 1-, 2-, 4- and 7-month-old pigs. The expression of sDab1 and sFyn-B was calculated as a percentage of GAPDH mRNA level in parallel, differences in groups were relative to the highest expression phase (set as 100%) in each group (*p < 0.05, **p < 0.01, ***p < 0.005; ^#p < 0.05, ^##p < 0.01, ^###p < 0.005).

Figure 4

Nucleocytoplasmic shuttling of sDab1 isoforms in IBRS2 cells. sDab1-GFP or sDab1-Li-GFP was transiently expressed in IBRS2 cells, and 24 hours after transfection, the cells were treated with methanol (vehicle control) (A or C) or 20 ng/ml LMB (B or D), and after 12 hours of treatment, the cells were fixed and counterstained with DAPI to visualize the nucleus. Scale bars: 20 μm.

Figure 5

Co-localization of sDab1 isoforms and sFyn-B in transfected primary neurons. A: sDab1 -GFP and sFyn-B-DsRed were co-transfected into DIV6 primary hippocampal neurons. B: sDab1-Li-GFP and sFyn-B-DsRed were co-transfected into DIV6 primary hippocampal neurons. 24 hours after transfection, the cells were fixed and counterstained with DAPI (blue) for nuclei. Scale bars: 20 μm.

Figure 6

Phosphorylation of sDab1 and sDab1-Li by sFyn-B in transfected Cos1 cells in vitro. A: sDab1-GFP or sDab1-Li-GFP was transfected to Cos1 cells along with sFyn-B-GFP. 24 hours after transfection, cells were lysed and analyzed by western blotting using 4G10, anti-Dab1, anti-Fyn and anti-Tubulin. B: Lanes 1-4: sDab1-GFP, sDab1-Li-GFP, mDab1-wt (murine wild-type Dab1 as a positive control) or mDab1-5F (murine mutant form of Dab1 carrying phenylalanine substitutions at residues Y185F, Y198F, Y200F, Y220F and Y232F as a negative control) were expressed in Cos1 cells along with porcine wild-type Fyn-B. Lanes 5-8: PP2, a specific inhibitor of SFKs, was added to the same set of co-transfections as in lanes 1-4. Lanes 9-12: sDab1, sDab1-Li, mDab1-wt or mDab1-5F was separately transfected to Cos1 cells without any exogenous Fyn. 24 hours after transfection, cells were lysed and analyzed by Western blotting using anti Dab1-pY185, anti Dab1-pY198/200 and anti Dab1-pY232. The membranes were also probed with anti-Dab1, anti-Fyn and anti-Tubulin antibodies to confirm protein loading. At least three blots were analyzed for these experiments.