ESSENTIAL ROLE OF PROTEIN-PHOSPHORYLATION IN NUCLEAR TRANSPORT

We have investigated a possible role for protein phosphorylation in nu clear transport in semi-intact cells, prepared by digitonin permeabili zation of rat F-111 fibroblasts. Treatment of semi-intact cells with a lkaline phosphatase abolished the import of nuclear transport substrat es, namely, signal peptide-albumin conjugates, as well as their signal -dependent binding at the nuclear pores, but did not affect the morpho logy of the cells, in particular their cytoskeletal network. Authentic transport and functional binding of the karyophilic protein at the nu clear envelope could be restored by incubation of phosphatase-treated cells with cytosol enriched in protein kinase C or with purified prote in kinase A (catalytic subunit). Restoration of transport was blocked by specific inhibitors of these kinases. Since the protein phosphoryla tion required for nuclear transport appeared to be a reasonably stable modification, characterization of the phosphorylated proteins was att empted in kinase reactions with radiolabeled ATP. Two proteins of 60-6 2 kDa were the predominant substrates phosphorylated by both protein k inase C and protein kinase A under conditions wherein nuclear transpor t was restored. Our results suggest a requirement for phosphorylation of one or more proteins for binding of a karyophilic protein at the nu clear envelope. (C) 1995 Academic Press,Inc.