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Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2
1Nutritional Sciences Program, University of Arizona, Tucson.
Abstract:
To investigate the effect of Mn2+ on pancreatic protein phosphorylation, we incubated rat pancreatic cytosol in Tris buffer (pH 7.5) with [gamma-32P]ATP. Analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography revealed a single protein (p98), with an Mr of 98,000 and a pI of 6.4-6.5, which was phosphorylated in a dose-dependent manner by Mn2+. A threshold effect was observed at 35 microM, and maximal effect at 1.1 mM Mn2+. Ca2+ and calmodulin (CaM) did not cause p98 phosphorylation, but Mg2+ (10 mM) caused faint non-specific phosphorylation of p98. Ca2+ (0.03-3 mM) and CaM (1-10 micrograms/ml) significantly enhanced, whereas trifluoperazine (TFP) and Mg2+ inhibited Mn(2+)-stimulated p98 phosphorylation. Under the above incubation conditions, Mn(2+)-stimulated protein phosphorylation of p98 was also observed in isolated pancreatic acini, but not in cytosols from liver or kidney. Partial purification of p98 and amino acid sequencing of the protein band corresponding to p98 indicated complete sequence homology with rat elongation factor 2 (EF-2). Furthermore, the combination of Ca2+, Mg2+ and CaM, which is known to induce the phosphorylation of EF-2, mimicked the actions of Mn2+. Inasmuch as EF-2 is the major substrate for CaM-dependent protein kinase III (CaM-PK III), these studies suggest that in the pancreatic acinar cell Mn2+/CaM protein kinase activity is mediated via CaM-PK III and the Mn2+ participates in the regulation of this enzyme in the pancreas.
Insights
Manganese (Mn2+) stimulates phosphorylation of a protein in pancreatic cells, identified as elongation factor 2 (EF-2). This suggests Mn2+ regulates EF-2 activity via CaM-PK III in the pancreas.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Protein phosphorylation is a key regulatory mechanism in cellular processes.
- Manganese (Mn2+) is a divalent cation with known roles in enzyme activity.
- The pancreas plays a vital role in digestion and endocrine functions, involving complex signaling pathways.
Purpose of the Study:
- To investigate the specific effects of Mn2+ on protein phosphorylation in pancreatic cells.
- To identify the protein(s) affected by Mn2+-induced phosphorylation.
- To elucidate the signaling pathway involved in Mn2+-mediated pancreatic protein phosphorylation.
Main Methods:
- Incubation of rat pancreatic cytosol and isolated acini with [gamma-32P]ATP and varying concentrations of Mn2+.
- Analysis of protein phosphorylation using SDS-PAGE and autoradiography.
- Partial purification, amino acid sequencing, and homology analysis of the phosphorylated protein.
Main Results:
- Mn2+ dose-dependently phosphorylated a specific protein (p98) in pancreatic cytosol and acini, with effects observed between 35 microM and 1.1 mM.
- The phosphorylated protein p98 was identified as rat elongation factor 2 (EF-2).
- Calcium (Ca2+), calmodulin (CaM), and Mg2+ modulated Mn2+-stimulated EF-2 phosphorylation, with Ca2+/CaM enhancing and trifluoperazine/Mg2+ inhibiting the process.
Conclusions:
- Mn2+ directly stimulates the phosphorylation of elongation factor 2 (EF-2) in pancreatic acinar cells.
- The observed Mn2+-stimulated EF-2 phosphorylation is mediated by CaM-PK III.
- These findings suggest a regulatory role for Mn2+ in pancreatic cell function through the modulation of EF-2 activity via CaM-PK III.