Essential role of protein phosphorylation in nuclear transport.
We have investigated a possible role for protein phosphorylation in nuclear transport in semi-intact cells, prepared by digitonin permeabilization of rat F-111 fibroblasts. Treatment of semi-intact cells with alkaline phosphatase abolished the import of nuclear transport substrates, namely, signal peptide-albumin conjugates, as well as their signal-dependent binding at the nuclear pores, but did not affect the morphology of the cells, in particular their cytoskeletal network. Authentic transport and functional binding of the karyophilic protein at the nuclear envelope could be restored by incubation of phosphatase-treated cells with cytosol enriched in protein kinase C or with purified protein kinase A (catalytic subunit). Restoration of transport was blocked by specific inhibitors of these kinases. Since the protein phosphorylation required for nuclear transport appeared to be a reasonably stable modification, characterization of the phosphorylated proteins was attempted in kinase reactions with radiolabeled ATP. Two proteins of 60-62 kDa were the predominant substrates phosphorylated by both protein kinase C and protein kinase A under conditions wherein nuclear transport was restored. Our results suggest a requirement for phosphorylation of one or more proteins for binding of a karyophilic protein at the nuclear envelope.
Mishra
K
K
Centre for Cellular and Molecular Biology, Hyderabad, India.
Parnaik
V K
VK
D000418:Albumins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003599:Cytoskeleton; D004072:Digitonin; D005347:Fibroblasts / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D056489:Nucleoplasmins; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D051381:Rats
D000418:Albumins; D000957:Antigens, Viral, Tumor; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010455:Peptides; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D004072:Digitonin