The extracellular signals controlling gene expression must ultimately be communicated to the nucleus. Nuclear transport plays a key role in regulating the levels and activities of transcription factors, nuclear kinases, steroid hormone receptors and replication factors. The nuclear pore complex (NPC) facilitates the movement of mRNA and proteins across the nuclear envelope. Previously, we described a family of phosphorylated glycoproteins which are components of the NPC. The major nuclear pore protein, p62, was cloned and expressed as a recombinant protein in E. coli and baculovirus. Sites of phosphorylation and glycosylation in p62 were mapped to the central Ser/Thr rich region of the molecule. Interestingly, changes in the degree of phosphorylation and glycosylation oscillated with the cell cycle. Phosphorylation reached a maximum during M phase; glycosylation peaked during S phase. This reciprocal regulation of phosphorylation and glycosylation may be associated with assembly and disassembly of NPC during the cell cycle. Using digitonin permeabilized cultured cells, we developed means of study nuclear and nucleolar transport of fluorescent conjugates bearing the appropriate localization signal. Such conjugates concentrated in the nucleus and nucleolus, respectively, when supplied with a cytosolic fraction and ATP. Nuclear transport was strongly activated by addition of Ca+2 or GTP to depleted cytosol. IP3 and calcium chelators inhibited transport; GTP-gamma-S also inhibited nuclear transport. The responsiveness of nuclear transport to Ca+2 and GTP suggests that nuclear transport may be coordinately regulated with other signal transduction pathways during cellular activation. We have extended these studies using patch-clamp electrophysiology to examine regulated ion flow through the NE of cardiac myocytes. We detected large conductance ion channel activity at the NE resembling that of gap junction channels. Activation of these channels required cytosolic factors. The channels were blocked by GTP-gamma-S, wheat germ agglutinin and nuclear pore-specific antibodies. Thus the large conductance nuclear ion channel activity is likely to be intrinsic to nuclear pores.
