Differential effect of insulin and epidermal growth factor on the mRNA translocation system and transport of specific poly(A+) mRNA and poly(A-) mRNA in isolated nuclei.
The efficiency of efflux of rapidly labeled poly(A)-containing mRNA from isolated rat liver nuclei was found to be modulated by insulin and epidermal growth factor (EGF) in a biphasic but opposite way. At physiological concentrations (10 pM insulin and 1 pM EGF), maximal stimulation of the transport rate by insulin (to 137%) and maximal inhibition by EGF (to 69%) were obtained; at higher concentrations (greater than 100 pM and greater than 10 pM, respectively), the amount of poly(A)-containing mRNA released into the postnuclear supernatant was nearly identical with the level found in untreated nuclei (= 100%). Using mRNA entrapped into closed nuclear envelope (NE) vesicles as a model system, it was found that the modulation of nuclear efflux of mRNA by the two growth factors occurs at the level of translocation through the nuclear pore. The NE nucleoside-triphosphatase (NTPase) activity, which is thought to mediate nucleocytoplasmic transport of at least some mRNAs, responded to insulin and EGF in the same manner as the mRNA transport rate. The increase in NTPase activity caused by insulin and the decrease in NTPase activity caused by EGF were found to be due to changes of the maximal catalytic rate; the Michaelis constant of the enzyme remained almost constant. Investigating the effect of the two growth factors on transport of specific mRNAs, poly(A)-containing actin mRNA was found to display the same alteration in efflux rate as rapidly labeled, total poly(A)-containing mRNA. In contrast, efflux of histone H4 mRNA, which lacks a 3'-poly(A) sequence, decreased in response to insulin and reached minimum levels at the same concentration at which maximum levels of actin mRNA transport rate were obtained. Studying the mechanism of action of insulin and EGF on NE mRNA translocation system, insulin was found to cause an enhancement of NE-associated phosphoprotein phosphatase activity, resulting in a dephosphorylation of the NE poly(A) binding site (= mRNA carrier) and, hence, in a decrease in its affinity to poly(A) [the poly(A) binding affinity of the poly(A)-recognizing mRNA carrier within the envelope is increased after phosphorylation]. EGF, on the other hand, stimulated the protein kinase, which phosphorylates the carrier, and, hence increased the NE poly(A) binding affinity. Because the stage of phosphorylation of the mRNA carrier (which is coupled with the NTPase within the intact NE structure) is inversely correlated with the activity of the NTPase, an enhancement of poly(A)-containing mRNA transport rate by insulin and an inhibition by EGF are observed.
Schröder
H C
HC
Institut für Physiologische Chemie, Universität, Mainz, Federal Republic of Germany.
Wenger
R
R
Ugarković
D
D
Friese
K
K
Bachmann
M
M
Müller
W E
WE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D009685:Nuclear Envelope / Q000201:enzymology; D043583:Nucleoside-Triphosphatase; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D012333:RNA, Messenger; D011061:Poly A; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase; D006152:Cyclic GMP