Abstract
Insulin induces the serine phosphorylation of the nucleolar protein nucleolin at subnanomolar concentrations in differentiated 3T3-442A cells. The stimulation is biphasic with phosphorylation reaching a maximum at 10 pM insulin and then declining to only 40% of basal levels at insulin concentrations of 1 microM. These changes are rapid, reaching half-maximal after 4 min and maximal after 15 min of incubation. The cell-permeable casein kinase II inhibitor 5,6-dichlorobenzimidazole-riboside prevents the insulin-stimulated phosphorylation of nucleolin suggesting that casein kinase II may mediate this effect of the hormone. Insulin-like growth factor 1 mimics the action of insulin on dephosphorylation of nucleolin at nanomolar concentrations suggesting that the latter effect may be mediated by insulin-like growth factor 1 receptors. Insulin treatment of 3T3-442A cells also results in a stimulation of RNA efflux from isolated, intact cell nuclei. The dose dependence of insulin-induced nucleolin phosphorylation and insulin-stimulated RNA efflux from intact cell nuclei are almost identical. Insulin induces an increase in the RNA efflux at subnanomolar concentrations in 3T3-442A adipocytes, while high (micromolar) concentrations of insulin inhibited the efflux of RNA. These data indicate that insulin regulates the phosphorylation/dephosphorylation of nucleolin, possibly via stimulation of casein kinase II, and this may play a role in regulation of the RNA efflux from nuclei.
Highlights
Insulininducestheserinephosphorylation of the effects involving enhanced nuclear activity (1,2)
Concentration Dependence of Insulin-induced Phosphorylation and Dephosphorylation of Nucleolin-Insulin induces the phosphorylation of several nuclear proteins in 3T3-442A cells which are able to bind to double-stranded DNA-cellulose.',*In theexperiment shown in Fig. 1,there is a marked insulin-induced phosphorylation of one 48-kDa and four 62-66-kDa proteins
The amountof phosphorylated nucleolin in the postnuclear Effect of Insulin-like Growth Factor I and a Casein Kinase supernatant was relatively minor 11 Inhibitor on Insulin-induced Nucleolin Phosphorylationcompared to that in nucleus and did not show significant Since 3T3-442A cells have IGF-1 receptors, and insulin is changesafterinsulinaddition.This low amount of phos- well-known to cross-reactwith these receptorsa t high, microphorylation was due to the low amount of non-nuclear nu- molar concentrations (28)w, e analyzed the effect of IGF-1 on cleolin as judged by immunoblotting of non-nuclear protein the phosphorylationof nucleolin
Summary
After the addition of insulin or other stimulants at concentrations specified in the individual experiments, the medium was removed, and the cells were scraped to an isolation buffer containing 20 mM HEPES, 1mM ATP, 5 mM MgCI,, 25 mM KC1, 2 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin, 50pg/ml leupeptin, 1 mM sodium vanadate, 1 mM sodium molybdate, 10 mM @-glycerophosphate, mM sodium pyrophosphate, 0.25 M sucrose, pH 7.4. Samples were rotated overnight at 4 "C, centrifuged in a microfuge, and the protein concentration of the supernatants was determined according to Bradford (19). Zmmunoblots-Non-radioactive nuclei were isolated, nucleolin was immunoprecipitated from aliquots of the nuclear fraction, and the postnuclear supernatant and thime munocomplexeswere purified and separated with SDS-PAGE as described above. After overnight incubation with a 1:200 dilution of rabbit anti-nucleolin serum a t 4 "C, the immunocomplexeswere visualizedby means of peroxidase-conjugated anti-rabbit antibodies and subsequent treatment with 4-chloro-lnaphthol and H,O,
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