Identification Of Proteins Interaction With Fbxl16 By Yeast Two-hybrid Technique

AIM: Fbxl16 is a novel protein which was found to be up-regulated after spinal cord injury. To search proteins that can interact with Fbxl16 to understand its function and roles in spinal cord injury and regeneration.METHODS: The EST sequence of scirr1 was cloned by RT-PCR. The PCR product was then sub-cloned into pSos plasmid to construct the vector pSos-scirr1. The fusion protein hSOS-Fbxl16 expressed by pSos-scirr1 served as bait protein, We used an improved yeast two-hybrid assay to screen rat brain cDNA library to search proteins that can interact with Fbxl16. Using yeast two-hybrid assay retransformation experiment, we verified of the interactions screened out at last.RESULTS: 11 proteins that are likely to interact with Fbxl16 were screened out after the construction of pSos-scirr1 by yeast two hybrid technique. The two proteins, protein kinase, cAMP-dependent, catalytic, alpha (Prkaca) and coatomer protein complex, subunit zeta 1(Copz1) were verified of proteins that can interact with Fbxl16.CONCLUSION: cAMP-PKA Pathway takes a great role in spinal cord injury. Regeneration of sensory axons within the injured spinal cord can be induced by intraganglionic cAMP elevation. Cyclic AMP elevates tubulin expression.Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury.Prkaca is one of the two main catalytic substances of PKA( cAMP-dependent protein kinase). The interaction of Fbxl16 with Prkaca helps to understand the relationship of cAMP-PKA Pathway and spinal cord injury. It suggests that Fbxl16 should play a role in the succeeding injury and/or repair processes of the injured spinal cord through cAMP-PKA Pathway.The interaction between Fbxl16 and Copz1, a subunit of coatomer protein complex(COP), which is involved in intra-Golgi transport and transport from the ER to the Golgi complex, suggests that Fbxl16 should be associated with intracellular transport.