Multiple phosphorylation of pp30, a rat brain polyribosomal protein, sensitive to polyamines and corticotropin.
A rat brain polyribosomal protein with an apparent Mr of 30 000, designated pp30, was further characterized. The protein was identified by its phosphorylation by an endogenous protein kinase sensitive to both corticotropin and spermine. Two-dimensional separation of a polyribosomal fraction was applied, combining non-equilibrium pH-gradient-gel electrophoresis in the first and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the second dimension. In this system, pp30 was separated into at least five defined phosphoprotein spots. Pulse-labelling with [gamma-32P]ATP followed by a chase for various time periods with excess unlabelled ATP resulted in a shift of the distribution of radioactivity and protein staining along the spots towards the anode. This suggests that the various spots of pp30 may represent multiple phosphorylation states. Limited proteolysis of the five spots with three different proteinases resulted in the same one-dimensional peptide maps with a given proteinase, indicating that all five spots represent different forms of a single phosphoprotein. Inhibition of the overall phosphorylation of pp30 by corticotropin or spermine was accompanied by a shift in the recovery of labelled phosphate towards spots nearer the cathode. Immunoblotting with monoclonal antibodies directed against ribosomal protein S6 stained only one band, a protein that had an apparent Mr of 34 000 and was clearly distinct from pp30.
D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007122:Immunoelectrophoresis; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism*; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology*
D010446:Peptide Fragments; D012269:Ribosomal Proteins; D013096:Spermine; D000324:Adrenocorticotropic Hormone