Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals.
Many important regulatory proteins, including cell cycle regulators and transcription factors, contain a phosphorylation site within or adjacent to a classic nuclear localization signal (NLS) sequence. Previous studies show that the nuclear localization of these cargoes can be regulated by phosphorylation at these sites. It was hypothesized that this phosphorylation regulates the nuclear import of NLS cargo proteins by modulating the interaction of the cargo with the classic nuclear transport receptor, importin alpha. In this study, we utilize in vitro solution binding assays and in vivo analyses to directly test this model. We demonstrate that mimicking phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for importin alpha. This decrease in cargo affinity for importin alpha correlates with a decrease in nuclear accumulation in vivo. Through these analyses, we show that the cell cycle-dependent nuclear import of the Saccharomyces cerevisiae transcription factor Swi6p correlates with a phosphorylation-dependent change in affinity for importin alpha. Furthermore, we present data using the SV40 NLS to suggest that this form of regulation can be utilized to artificially modulate the nuclear import of a cargo, which is usually constitutively targeted to the nucleus. This work defines one molecular mechanism for regulating nuclear import by the classic NLS-mediated transport pathway.
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA.
Michelle T
MT
Harreman
Trisha M
TM
Kline
Heidi G
HG
Milford
M Beth
MB
Harben
Alec E
AE
Hodel
Anita H
AH
Corbett
D000595:Amino Acid Sequence; D002453:Cell Cycle; D006706:Homeostasis; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D019913:Nuclear Localization Signals; D010446:Peptide Fragments; C075111:SWI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors