No abstract

Protein kinase activities that are able to phosphorylate specific ribosomal proteins have been found in a variety of eukaryotic systems (reviewed [l--3]). Such enzymes catalyze the incorporation of phosphoryl groups into the serine and threonine residues of those proteins [4-61. They are active as well in the in vitro pho~ph~~lation of exogenous protein substrates other than ~bos~~ proteins, According to their substrate spec~~c~ty, protein kinases can thus be classBed into two main groups: protein kinases that preferenti~ly p~~s~~~~late acidic proteins such as casein or phosvitiin, and protein kinases which instead are rather specific for basic proteins such as histones or protamin [7]. These two groups of enzymes can be further differentiated by their degree of stimulation of phosphorylation in the presence of cyclic AMP, their ability to utilize ATF or GTP as a ph~sPho~1 donor, and their relative activity in the presence of salts or or other effecters such as hide IS]. S&h distinct enzymes have been snorted to be bound to ribosomes in several systems in&ding horse thyroid glands [of, yeast [IO], mouse plasma cell tumors [Xl], bovine corpus luteum [12] and rabbit reticulocytes [7]. Furthermore, experiments carried out with rat liver have shown that membrane-free polysomes harbor simultaneously at least two different protein kinase activities: a histone kinase* type enzyme and a casein kinase-type enzyme [13], However, in neither of these cases, it has been described wbethei the substrate specificity of kinases found for exogenous protein substrates applies to the endoge~o~s phosphorylation of ribosomal proteins.In this work such a study was therefore undertaken in rat liver by estimating the extent of in vitro phosphorylation of specific ribosomal proteins catalyzed separately by each protein kinase activity associated with membrane-free polysomes. The results presented indicate mainly that basic and acidic ribosomal prateins are selectively ~h~sph~~lated by different protein ktnases, The method used for preparing membrane-free rat liver polysomes and the experimental procedure followed to treat them by various KC1 concentrations have been detailed in [ 14,15].The protein kinase activity of polysomes on exogenous substrates @stone and casein) was detested as in f 13 >f 4] by estima~g the ~~~~t of o~~pbos~~ate ~~co~~~a~e~ into profein at the expense of radioactive ATP, Acidic ribosomal proteins were extracted according to [IS] by suspending phosphorylated polysomes (200 &Q units) in 0.6 ml of a buffer containing 50 mM triethanolamine-HCl, at pH 7.4,80 mM KCI, 5 mM MgClzp 20 PM &mercaptoethanol and 0,6 ml absolute ethanol, The suspension was stirred for 15 mln at 4% then centrifuged for IO min at 5000 X g. The procedure was repeated twice and the su~rnat~t fractions containing the acidic proteins were added 3 times for 8 h each against 5 1 of f M

No abstract

Rat liver polysomes washed with 0.5-1.5 M KCl at 37 degrees C keep a constant protein kinase activity revealed only by auto-phosphorylation of ribosomal proteins. The enzyme catalyzes the transfer of the gamma-phosphate group from ATP to serine (75%) but also to threonine residues (25%). It is released when polysomes are dissociated into subunits using centrifugation through a sucrose gradient containing a high K+/Mg++ ratio. Its properties have been compared with those of the two other enzymatic activities which are, in contrast, washed out during salt treatment of polysomes. After release upon polysome dissociation, this third activity is able to phosphorylate histone II A. Protection of the enzyme in the polysome structure against salt treatment, suggests that it is located at the junction of the two subunits.