Heterodimerization with Jun Family Members Regulates c-Fos Nucleocytoplasmic Traffic

Harald Wodrich,Marc Piechaczyk,Tamara Salem,Cécile E Malnou,Frédérique Brockly,Isabelle Jariel-Encontre

doi:10.1074/jbc.m702833200

Abstract

c-Fos proto-oncoprotein forms AP-1 transcription complexes with heterodimerization partners such as c-Jun, JunB, and JunD. Thereby, it controls essential cell functions and exerts tumorigenic actions. The dynamics of c-Fos intracellular distribution is poorly understood. Hence, we have combined genetic, cell biology, and microscopic approaches to investigate this issue. In addition to a previously characterized basic nuclear localization signal (NLS) located within the central DNA-binding domain, we identified a second NLS within the c-Fos N-terminal region. This NLS is non-classic and its activity depends on transportin 1 in vivo. Under conditions of prominent nuclear localization, c-Fos can undergo nucleocytoplasmic shuttling through an active Crm-1 exportin-independent mechanism. Dimerization with the Jun proteins inhibits c-Fos nuclear exit. The strongest effect is observed with c-Jun probably in accordance with the relative stabilities of the different c-Fos:Jun dimers. Retrotransport inhibition is not caused by binding of dimers to DNA and, therefore, is not induced by indirect effects linked to activation of c-Fos target genes. Monomeric, but not dimeric, Jun proteins also shuttle actively. Thus, our work unveils a novel regulation operating on AP-1 by demonstrating that dimerization is crucial, not only for active transcription complex formation, but also for keeping them in the compartment where they exert their transcriptional function.

Highlights

The best-studied AP-1 components are the Fos (c-Fos, FosB, Fra-1, and Fra2) and Jun (c-Jun, JunB, and JunD) proteins that dimerize via a leucine zipper (LZ)4 domain and recognize their target DNA sequences owing to an adjacent, upstream basic DNA-binding domain (DBD) [6]
Our main results were as follows: (i) We found no evidence for an nuclear localization signals (NLS) located C-terminally of the DBD, i.e. neither within the LZ nor within the C-terminal domain (Fig. 2A), because neither LZ(c-Fos⌬163–196 mutant) nor C-terminal domain (c-Fos-(1– 196)) deletion detectably altered c-Fos nuclear localization (Fig. 2B)
Nuclear Import of c-Fos—Here, we have localized the second c-Fos NLS within the N-terminal domain and shown that the combined action of the two NLSs is required for quantitative c-Fos nuclear accumulation

Summary

EXPERIMENTAL PROCEDURES

Plasmids, Cloning, and Mutagenesis—Cloning and mutagenesis were performed using standard PCR-based methods into the cytomegalovirus promoter-based pcDNA3 expression vector (Invitrogen). 1 h later, freshly trypsinized HeLa cells, previously transfected with the pDsRed2-C1 plasmid for 16 h, were added in the presence of CHX. After another 2 h, i.e. time sufficient for cell spreading, coverslips were quickly rinsed in PBS (150 mM NaCl, 10 mM sodium phosphate, pH 7), covered with a 50% w/v solution of PEG for 2 min, again carefully washed with PBS, and, incubated at 37 °C for 1 h in fresh medium containing 10% serum and CHX. For analysis of ectopic wild-type and mutant c-Fos and c-Jun proteins as well as that of EGFP chimeras, HeLa cells were co-transfected with pDsRed2-C1 and the relevant expression vector(s). Tazi) was used to characterize the insoluble one (see Ref. 16)

RESULTS

Findings

DISCUSSION