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.