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Multiple phosphorylation of pp30, a rat brain polyribosomal protein, sensitive to polyamines and corticotropin
Abstract:
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.
Insights
This study characterizes pp30, a rat brain polyribosomal protein. Findings suggest pp30 exists in multiple phosphorylation states, regulated by corticotropin and spermine.
Area of Science:
- Neuroscience
- Molecular Biology
- Protein Biochemistry
Background:
- Polyribosomal proteins play crucial roles in protein synthesis regulation within neurons.
- Understanding protein phosphorylation is key to deciphering cellular signaling pathways.
Purpose of the Study:
- To further characterize a rat brain polyribosomal protein, pp30, focusing on its phosphorylation states and regulation.
- To investigate the relationship between pp30 and other known ribosomal proteins.
Main Methods:
- Two-dimensional gel electrophoresis (non-equilibrium pH-gradient-gel electrophoresis and SDS-PAGE) was used to separate pp30.
- Pulse-labeling with [gamma-32P]ATP and limited proteolysis were employed to analyze phosphorylation.
- Immunoblotting with monoclonal antibodies against ribosomal protein S6 was performed.
Main Results:
- pp30 was resolved into at least five distinct phosphoprotein spots, indicating multiple phosphorylation states.
- Radioactivity distribution shifts suggested dynamic phosphorylation changes regulated by corticotropin and spermine.
- Limited proteolysis confirmed all pp30 spots represent forms of a single phosphoprotein.
- pp30 was distinguished from ribosomal protein S6 (Mr 34,000).
Conclusions:
- Rat brain polyribosomal protein pp30 exhibits multiple phosphorylation states.
- Corticotropin and spermine modulate pp30 phosphorylation, suggesting a regulatory role in protein synthesis.
- pp30 is a distinct phosphoprotein, separate from ribosomal protein S6.