Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000-dalton protein phosphorylated in situ in response to secretagogues.

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Abstract
The endogenous phosphorylation of a protein with a MW of 29,000 was enhanced by various secretagogues in rat pancreatic and parotid lobules, the phosphorylation of this protein correlating both temporally and in a dose-dependent fashion with secretory protein discharge. A specific methodology was established to characterize this phosphoprotein. Once established, this 29,000-dalton phosphoprotein was then followed selectively and quantitatively throughout subcellular fractionation procedures. Analysis of 2-dimensional polyacrylamide gels demonstrated that proteins with similar mobilities (MW 29,000; pI > 8.4) were affected by cholecystokinin octapeptide and isoproterenol in rat pancreatic and parotid lobules, respectively, suggesting that the same 29,000-dalton phosphoprotein was covalently modified in both tissues. Cellular fractionation studies using differential velocity and sucrose density gradient centrifugation revealed that the 29,000-dalton phosphoprotein copurified with the rough microsomal fraction of pancreas and was highly enriched in ribosomal fractions of both pancreas and parotid. Electrophoresis in 2 dimensions confirmed that the 29,000-dalton polypeptide that was resolved directly from stimulated cells and from ribosomal fractions exhibited a common mobility, and apparent identity of the species was strongly suggested when the 29,000-dalton polypeptides from both sources were compared by peptide mapping following limited digestion with Staphylococcus aureus V8 protease. This phosphoprotein was tentatively identified as ribosomal protein S6 after analysis by pH 8.6/4.2 2-dimensional polyacrylamide gel electrophoresis.