Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures.
Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D001692:Biological Transport; D002645:Chickens; D003599:Cytoskeleton / Q000276:immunology / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009685:Nuclear Envelope / Q000201:enzymology / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D010703:Phorbol Esters / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D010703:Phorbol Esters; D012333:RNA, Messenger; D011061:Poly A; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase