Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation.
Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002458:Cell Fractionation; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002147:Calmodulin; D008565:Membrane Proteins; D013439:Sulfhydryl Reagents; D000242:Cyclic AMP; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D006152:Cyclic GMP