Nuclear Import of c-Jun Is Mediated by Multiple Transport Receptors

Abstract

c-Jun and c-Fos are major components of the transcriptional complex AP-1. Here, we investigate the nuclear import pathway(s) of the transcription factor c-Jun. c-Jun bound specifically to the nuclear import receptors importin ␤, transportin, importin 5, importin 7, importin 9, and importin 13. In digitoninpermeabilized cells, importin ␤, transportin, importin 7, and importin 9 promoted efficient import of c-Jun into the nucleus. Importin ␣, by contrast, inhibited nuclear import of c-Jun in vitro. A single basic region preceding the leucine zipper of c-Jun functions as a nuclear localization signal (NLS) and was required for interaction with all tested import receptors. In vivo, nuclear import of a c-Jun reporter protein lacking the leucine zipper strictly depended on this NLS. In a leucine zipper-dependent manner, c-Jun with mutations in its NLS was still imported into the nucleus in a complex with endogenous leucine zipper proteins or, for example, with cotransfected c-Fos. Together, these results explain the highly efficient nuclear import of the transcription factor c-Jun.Nuclear transport of proteins is mediated by soluble receptor molecules belonging to the importin ␤ superfamily. These proteins, collectively referred to as karyopherins or, depending on the direction of transport, importins and exportins, are characterized by tandem copies of HEAT repeats (for Huntingtin, elongation factor 3, protein phosphatase 2A, TOR1) that provide an interaction surface for many different binding partners. HEAT repeats are ϳ40 amino acids in length and adopt a helixturn-helix conformation with anti-parallel helices. The 18 -20 HEAT repeats result in a superhelical overall structure of the transport receptors with a high degree of flexibility, facilitating the binding to a large variety of different proteins (for review, see Refs. 1-2). Common interaction partners of all karyopherins are the small GTP-binding protein Ran as well as certain nucleoporins, components of the nuclear pore complex. In nuclear export, RanGTP is an integral component of the transport complex. In nuclear import, by contrast, RanGTP functions in the dissociation process of import complexes in the nucleus (3), as binding of cargo molecules and RanGTP to importins is considered to be mutually exclusive. A high concentration of nuclear RanGTP, as required for the formation of export complexes and dissociation of import complexes, results from the activity of the chromatin-associated guanosine nucleotide exchange factor for Ran, RCC1. In the cytoplasm, on the other hand, the GTPase-activating protein RanGAP together with the Ran-binding protein RanBP1 promote GTP hydrolysis on Ran. RanGDP is then imported into the nucleus by a dedicated transport factor, NTF2 (for review, see Refs. 4 and 5).Importins recognize their cargo molecules via certain nuclear localization signals (NLSs).2 The "classic" NLS is characterized by short stretches of basic amino acids, typically lysine residues, occurring either as a single or a bipartite motif. Both motifs interac...

A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1

“…Indeed, even though it is assumed that each epitope can accommodate substantial sequence diversity, the limits are still vague. The fact that some characterized Trn1 cargos lack a PY-NLS suggests that the actual variability is larger than currently conceived (12)(13)(14)(15). We believe that more bimodular NLSs, such as in ADAR1, will be found in the future and that our study will help identifying them.…”

“…All mutant proteins were checked to be properly folded by running ( 15 N, 1 H)-HSQC spectra. Proteins were overexpressed in BL21(DE3) Codon-plus (RIL) cells in either LB media or M9 minimal media supplemented with 15 NH 4 Cl and 13 C-labeled glucose. The cells were grown at 37°C to OD600 ∼0.4, cooled down at 30°C, and induced at OD600 ∼0.6 by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM.…”

“…These principles can be described as follows: a peptide segment of 15-30 residues with intrinsic structural disorder, an overall basic character, and some weakly conserved sequence motifs, including a relatively conserved proline-tyrosine (PY) dipeptide, leading to the name PY-NLS for this class of signals (8). However, many characterized Trn1 cargoes do not contain such a PY-NLS (4,(12)(13)(14)(15). Therefore, Trn1 interacts not only with the well-characterized PY-NLSs, but at least with another class of NLS, the recognition of which is far less understood.…”

Active maintenance of nuclear actin by importin 9 supports transcription

“…6B). Ipo9 has other cargos besides actin, and it has been implicated in the nuclear import of histones (45) and c-Jun (46), but the decreased transcription appears actin dependent as the delivery of actin into the nucleus via another import pathway significantly restored the levels of the nascent mRNA ( Fig. 6 B and C).…”

Increased expression of c-Jun in nonalcoholic fatty liver disease

“…46 AP-1 is a homo-or heterodimer consisting of proteins belonging to the c-Jun, c-Fos, ATF and JDP families, 47 with c-Jun being the best-characterized AP-1 component. The nuclear import of c-Jun is mediated by multiple mechanisms 48,49 and nuclear c-Jun levels correlate with AP-1 target gene activity. 49 This further increases c-Jun protein abundance, as the jun proto-oncogene itself is activated by AP-1 in the manner of a positive auto-regulatory loop.…”