A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.
A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.
D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000097:blood*; D012270:Ribosomes / Q000378:metabolism
D012269:Ribosomal Proteins; D000242:Cyclic AMP; D011494:Protein Kinases