A nuclear localization signal is required for the nuclear translocation of Fign and its microtubule‑severing function

Abstract

It is commonly known that the specific function of a given ATPase associated with diverse cellular activities protein (i.e., a member of the AAA superfamily of proteins) depends primarily on its subcellular location. The microtubule‑severing protein fidgetin (Fign) possesses a nuclear localization signal (NLS) that facilitates its translocation to the nucleus, where its assembly is finalized; here, Fign contributes to the regulation of microtubule configuration by cutting and trimming microtubule polymers. In the present study, Fign was found to be a nuclear protein, whose N‑terminal sequence (SSLKRKAFYM; residues 314‑323) acts as an NLS. Following substitution (KR to NN; 317‑318) or deletion (NT; 314‑323) mutations within the NLS, Fign, which is predominantly expressed in the nucleus, was found to reside in the cytoplasm of transfected cells. Furthermore, Fign was found to have an essential role in microtubule severing by preferentially targeting highly‑tyrosinated microtubules (tyr‑MTs). Mutation of the Fign NLS did not affect its microtubule‑severing function or the cleavage of tyr‑MTs, but did affect the cellular distribution of the Fign protein itself. Taken altogether, an NLS for Fign was identified, and it was demonstrated that the basic amino acids K317 and R318 are necessary for regulating its entry into the nucleus, whereas an increase in Fign in the cytosol due to mutations of the NLS did not affect its cleavage function.

Keywords: fidgetin; nuclear localization signal; microtubule severing; tyrosinated modification.

Figure 1.

Construction of the Fign-GFP plasmid and subcellular localization of Fign primarily expressed in the nucleus. (A) Agarose gel electrophoresis of PEGFP-N1 universal primers. Lane 1, plasmid-positive colony of Fign-GFP; lanes 2–3, plasmid-negative colony; lane M, 2 kb - DNA ladder marker. (B) Fign protein expression detected by western blotting. (C) Subcellular localization of Fign-GFP in 293T cells. Fign, fidgetin.

Figure 2.

Schematic representation of the putative NLS and subcellular localization of Fign-mNLS-GFP and Fign-ΔNLS-GFP proteins. (A) Schematic representation of Fign protein and the putative NLS (amino acids 314–323). (B) Schematic representation of the NLS and its mutants. Fign-mNLS-GFP, KR (317–318) was replaced by NN; Fign-ΔNLS-GFP, the NLS was deleted. (C) Agarose gel electrophoresis of Fign and its fragments. Lane 4, Fign-mNLS-GFP colony PCR; lane 8, Fign-ΔNLS-GFP colony PCR; lanes 1 and 2, N-terminal fragments and C-terminal of Fign-mNLS-GFP; lanes 5 and 6, N-terminal and C-terminal fragments of Fign-ΔNLS-GFP; lanes 3 and 7, mNLS and ΔNLS fragments of Fign. (D) Expression of mutant proteins was confirmed by western blotting. (E) Ratio of the average GFP signals in the nucleus and whole-cell. The cell and nuclear morphology were delineated under white light and DAPI conditions, respectively, and the gray value and area of the nuclei and intact cells were calculated by Image-Pro Plus 7.0 software. Nucleus signal ratio=nuclear grayscale mean/cell grayscale mean. (F) Fign and its mutant proteins expressed in HeLa cells. *P<0.05 vs. Control group. ^#P<0.05 vs. Control group. Fign, fidgetin; NLS, nuclear localization signal.

Figure 3.

Fign and its mutants do not sever total microtubules. (A) Total microtubules in cells transfected with Fign and its mutants. (B) Quantification of the relative fluorescence intensity of α-tubulin protein using Image-Pro Plus 7.0 software; compared to the GFP group, n=12. (C) The expression of mutant protein was confirmed by western blotting. (D) Representative western blotting result showing that there are no change of α-tubulin between Fign and its mutant. (E) Statistical data showing no change of α-tubulin protein after Fign overexpression. The data are shown as mean ± SE. Fign, fidgetin; NLS, nuclear localization signal.

Figure 4.

Fign and its mutants sever tyr-tubulin. (A) Tyr-tubulin in cells transfected with Fign and its mutants. (B) Quantification of the relative fluorescence intensity of tyr-tubulin protein using Image-Pro Plus 7.0 software; compared to the GFP group, n=12. (C) Representative western blotting result showing that Fign and its mutant overexpression cultures have a lower ratio of tyr-tubulin to α-tubulin than GFP cultures. (D) Statistical data showing a decrease of tyr-tubulin protein after Fign and its mutants were overexpressed. The data are shown as the mean ± SE. *P<0.05 vs. Control group. Fign, fidgetin; tyr-, tyrosine; NLS, nuclear localization signal.