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Abstract

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S^111/112. In this study purified p34^cdc2 kinase was used to phosphorylate T antigen proteins specifically at T¹²⁴ and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T¹²⁴ phosphorylation, which could be functionally simulated by a T-to-D¹²⁴ substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T¹²⁴ and S^111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T¹²⁴ phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T¹²⁴ phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.