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Identification of alternatively spliced Dab1 and Fyn isoforms in pig.

Long H, Bock HH, Lei T, Chai X, Yuan J, Herz J, Frotscher M, Yang Z - BMC Neurosci (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China.

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.

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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).
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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).

Mentions: Apart from regulation of neuronal migration during embryonic development, Reelin-Dab1-SFKs signaling is also relevant for the modulation of synaptic plasticity and learning in the adult mouse brain[27,28]. Hence, it is important to test temporal expression patterns of sDab1 and sFyn-B in the postnatal pig brain. Cerebral cortex and cerebellum of 1-, 2-, 4-, 7-month-old Meishan pigs were chosen to investigate the temporal expression profiles of sDab1 and sFyn-B. Of note, they exhibited similar expression profiles. The expression of sDab1 and sFyn-B reached the highest level in the cerebral cortex at the age of 2 months (p < 0.005/0.01) (Figure 3-A, B; black columns) and at the age of 4 months (p < 0.01/0.05) in the cerebellum (Figure 3-A, B; white columns), respectively, and declined afterwards.


Identification of alternatively spliced Dab1 and Fyn isoforms in pig.

Long H, Bock HH, Lei T, Chai X, Yuan J, Herz J, Frotscher M, Yang Z - BMC Neurosci (2011)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3044655&req=5

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).
Mentions: Apart from regulation of neuronal migration during embryonic development, Reelin-Dab1-SFKs signaling is also relevant for the modulation of synaptic plasticity and learning in the adult mouse brain[27,28]. Hence, it is important to test temporal expression patterns of sDab1 and sFyn-B in the postnatal pig brain. Cerebral cortex and cerebellum of 1-, 2-, 4-, 7-month-old Meishan pigs were chosen to investigate the temporal expression profiles of sDab1 and sFyn-B. Of note, they exhibited similar expression profiles. The expression of sDab1 and sFyn-B reached the highest level in the cerebral cortex at the age of 2 months (p < 0.005/0.01) (Figure 3-A, B; black columns) and at the age of 4 months (p < 0.01/0.05) in the cerebellum (Figure 3-A, B; white columns), respectively, and declined afterwards.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China.

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.

Show MeSH