Overlapping binding sites for importin β1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization

A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Imp beta 1 (importin beta 1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Imp beta 1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Imp beta 1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Imp beta 1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Imp beta 1 for binding to Gli1, through the fact that the sites for SuFu and Imp beta 1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Imp beta 1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.