IDENTIFICATION OF A NUCLEAR-LOCALIZATION SEQUENCE WITHIN THE STRUCTURE OF THE HUMAN INTERLEUKIN-1-ALPHA PRECURSOR

Recent studies have suggested that the signal peptide-less cytokine, i nterleukin (IL)-1alpha, may play a role as an intracellular regulator of human endothelial cell proliferation in vitro (Garfinkel, S., Haine s, D. S., Brown, S., Wessendorf, J., Gillespie, D. H., and Maciag, T. (1992) J. Biol. Chem. 267, 24375-24378). In order to determine the int racellular locale of the IL-1alpha precursor, we fused the open readin g frame of the IL-1alpha precursor to the reporter gene beta-galactosi dase (Gal) and studied the cellular distribution of the chimera in NIH 3T3 cells after transfection. Immunological and enzymatic analysis de monstrated that the IL-1alpha:beta-Gal fusion protein was associated w ith the nucleus. To further define the region responsible for this act ivity, we ligated the mature form of IL-1alpha (IL-1alpha113-271) and the IL-1alpha precursor domain (IL-1alpha1-112) to beta-Gal. Analysis of the intracellular distribution of these chimeric polypeptides follo wing transfection demonstrated a differential distribution of IL-1alph a1-112:beta-Gal in the nucleus and IL-1alpha113-271:beta-Gal in the cy tosol. Because the IL-1alpha precursor domain contains a sequence that resembles a nuclear translocation signal (KVLKKRRL, residues 79-86), we prepared an IL-1alpha precursor point mutant in which Lys82 was rep laced by Glu. Transfection of NIH 3T3 cells with the IL-1alpha precurs or point mutant (IL-1alpha1-271 Glu82:beta-Gal) resulted in a signific ant reduction in the ability of the IL-1alpha precursor to associate w ith the nucleus and similar data were obtained as a result of Lys82 mu tagenesis in the IL-1alpha precursor domain (IL-1alpha1-112 Glu82:beta -Gal). These data suggest that the IL-1alpha precursor contains a func tional nuclear localization sequence within the structure of the precu rsor domain and Lys82 is critical for its function.