CYCLIN-DEPENDENT KINASE SITE-REGULATED SIGNAL-DEPENDENT NUCLEAR-LOCALIZATION OF THE SWI5 YEAST TRANSCRIPTION FACTOR IN MAMMALIAN-CELLS

Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SW15 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G(1). beta-galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro-Arg-Lys(655)) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G(1) or G(2)/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser micros copy, Cell cycle dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser(646) and Ser(664)) to alanine resulted in constitutive nuclear localization, In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold, We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.