THE CAMP-DEPENDENT PROTEIN-KINASE SITE (SER(312)) ENHANCES DORSAL NUCLEAR IMPORT THROUGH FACILITATING NUCLEAR-LOCALIZATION SEQUENCE IMPORTIN INTERACTION/

Control over the nuclear import of transcription factors (TFs) represe nts a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila TF Dorsal shares wi th other rel TF family members the fact that it contains a phosphoryla tion site for the cAMP-dependent protein kinase (PKA) 22 amino acids N -terminal to the nuclear localization signal (NLS) at amino acids 335- 340. This study examines for the first time the nuclear import kinetic s of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro . Nuclear uptake was found to be not only NLS-dependent, but also stro ngly dependent on the PKA site, whereby substitution of Ser(312) by ei ther Ala or Glu using site-directed mutagenesis severely reduced nucle ar accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/ 97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dor sal NLS and PKA site constitute a phosphorylation-regulated NLS essent ial to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS reco gnition by importin.