Effect of site-directed mutagenesis on neurofilament dynamics.

摘要

Neurofilaments (NFs) are an essential part of the neuronal cytoskeleton that stabilizes the axon. NFs are capable of undergoing transport along the axon and forming a stationary phase (crosslinked-NFs that do not undergo transport themselves). Formation of the stationary phase requires the association of NF-NF that is regulated by NF phosphorylation. Phosphorylation mainly occurs in the 'C-terminus' of the NF, which allows it to extend laterally away from the NF backbone and form crossbridges between NFs and other NFs. This competes with the axonal transport as hyper-phosphorylated NFs undergo transport at a slower rate as compared to non-phosphorylated NFs. Most of the C-terminal phosphorylation occurs in the KSP consensus sites, which is the main regulatory factor in NF transport and NF bundling, but our study focuses on a single phosphorylation site in the E-segment of the C-terminus (Ser-493). Phosphorylation of Ser-493 begins at the same time point in the development of the axon as the phosphorylation of KSP sites. In order to study the role of Ser-493 on NF dynamics, we mutated Ser-493 to alanine in the heavy-chain Neurofilament (NFH) to mimic the constitutively non-phosphorylated state of this site. Another derivative of NFH was constructed by coupling the Ser-493 to alanine mutation with the truncation of the sidearm that is responsible for the formation of the bundles. Differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged full length NFH, NFH in which Ser-493 was mutated to alanine (S493A), NFH lacking the distal-most 187 amino acids of the sidearm (HΔ187) or both (HΔ187+S493A). All constructs co-assembled with the endogenous NF network and underwent transport at similar rates. Overexpression of GSK3β and CKIα did not alter the transport rate but increased the bundling of full-length NFH. The bundling was not induced in HΔ187, S493A and HΔ187+S493A with the overexpression of GSK3β and CKIα; however, the transport was reduced by overexpression of GSK3β in HΔ187 and overexpression of CKIα in S493A and HΔ187+S493A. Inhibition of GSK3β and CKIα did not alter the axonal transport but inhibition of GSK3β decreased the bundling of full-length NFH. Inhibition of GSK3β increased transport in HΔ18, while inhibition of CKIα increased transport of S493A and HΔ187+S493A. Blot analysis of the fractions enriched in the bundled NFs and the individual NFs showed that the bundled NFs displayed an increase in phosphorylated epitopes seen early in the development in all the constructs indicating that bundled NFs had a high number of phosphorylated NFs that are capable of undergoing transport. Upon inhibition of GSK3β, the non-phosphorylated epitopes increased in the bundled NFs for all the constructs, while with the inhibition of CKIα an increase was observed in the early-phosphorylated epitopes in NFH cultures and in non-phosphorylated epitopes for S493A and HΔ187+S493A in the bundled NFs. These findings show that the phosphorylation of Ser-493 inhibits the transport of NFs along the axon, suggesting that Ser-493 plays a regulatory role in the axonal transport. Ser-493 also plays a role in regulating the events that in turn regulate the phosphorylation of the C-terminal by other kinases thereby controlling the bundling of NFs.;Keywords: neurofilament, phosphorylation, axonal transport, Ser-493. NF-NF association.