Cloning, Expression And Regulation Research On Cytoskeletal Rearrangment Of Porcine Dab1 And Fyn Genes

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. Fyn, a member of the Src tyrosine kinase family (SFK), is involved in phosphorylation of Dab1 and then tranduce Reelin signaling. As a non-repetor tyrosine kinase, Fyn participates in different cellular signaling pathway and regulates cell migration, proliferation, differentiation, motility and growth. These processes are mediated by signaling pathways that cause cytoskeletal rearrangements. However, the molecular mechanisms of these Fyn-induced rearrangements are not yet fully understood.In the present study, by using RT-PCR, point mutation, overexpression, immunohistochemistry and western blotting, we cloned porcine Dab1 and Fyn isoforms and studied on their interaction and functions on regulating cytoskeleton rearrangement, our results were following:1. 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), then analysed the splicing pattern of both genes.2. Radiation hybrid mapping localized porcine Dab1(sDab1) and Fyn (sFyn) to chromosomes 6q31-35 and 1p13, respectively.3. Real-time quantitative RT-PCR (qRT-PCR) demonstrated that different isoforms of Dab1 and Fyn have tissue-specific expression patterns, sDab1 is highly expressed in neuro-system, while sDab1-Li is an isoform that is highly expressed in peripheral organs; sFyn-B is highly expressed in neuro-system, while sFyn-T is highly expressed in immuno organs. sDab1 and sFyn-B display similar temporal expression characteristics in the developing porcine cerebral cortex and cerebellum.4. Both sDab1 isoforms function as nucleocytoplasmic shuttling proteins.5. sFyn-B phosphorylates sDab1 at tyrosyl residues (Tyr) 185,198/200 and 232, whereas sDab1-Li was phosphorylated at Tyr 185 and Tyr 197 (corresponding to Tyr 232 in sDab1) in vitro. 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. 6. Overexpression of porcine Fyn (wt-Fyn) in different cells induces extended changes in cell shape as well as filopodia and lamellipodia formation. We examined the localization of Fyn mutants lacking different functional domains and analyzed the effects of these mutations on the rearrangement of the actin cytoskeleton in IBRS2 cells. During this process, the existence of SH2 and SH3 domains is crucial for the subcellular localization of Fyn, but not for its activation; the correct modification of Fyn protein ensure its activation.7. Fyn-induced formation of filopodia and lamellipodia might depend on the ability of Fyn to bind to focal adhesion sites, which in turn leads to the subsequent phosphorylation of focal adhesion kinase (FAK) at Tyr 861 and Tyr 925, thus resulting in the rearrangement of the actin cytoskeleton. Reelin enhanced phosphorylation of FAK at Tyr 861 is SFK-dependent and this effect can be blocked by PP2. Hence, Reelin might regulate spine morphology via Fyn-FAK pathway.8. Fyn promotes dendritic branching in primary neurons and increases the dendritic spine density of pyramidal neurons in hippocampal slices. The kinase activity and SH3 domain are necessary for Fyn-regulated enhancement of dendritic spine density.