Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using dig itonin-permeabilized cell assays to measure nuclear import and export, we f ound that the phosphatase inhibitors okadaic acid and microcystin inhibit t ransport mediated by the import receptors importin beta and transportin, bu t not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad speci ficity protein kinase inhibitor staurosporine, indicate that transport inhi bition is due to elevated phosphorylation of a component of the nuclear tra nsport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive compone nt of the nuclear transport machinery also is present in permeabilized cell s and is most likely a component of the nuclear pore complex. Substrate bin ding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphat ase inhibitors, suggesting that transport inhibition by protein phosphoryla tion does not involve these steps. These results suggest that cells have me chanisms to negatively regulate entire nuclear transport pathways, thus pro viding a means to globally control cellular activity through effects on nuc leocytoplasmic trafficking.