Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2.
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.
Knight
S A
SA
Nutritional Sciences Program, University of Arizona, Tucson.
Kohr
W
W
Korc
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010743:Phospholipids; D010750:Phosphoproteins; D008345:Manganese; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium