Manganese-stimulated protein phosphorylation in the rat pancreas.

Abstract

Mn²⁺ has been proposed to be a regulator of pancreatic function. To investigate the effect of Mn²⁺ on pancreatic protein phosphorylation, we incubated rat pancreatic cytosol in Tris buffer (pH 7.5) with [γ-³²P] ATP. Analysis using SDS-PAGE and autoradiography revealed a single protein (p98), with a Mr of 98 kDa and a pI of 6.4 to 6.5, which was phosphorylated in a dose-dependent manner in the presence of Mn²⁺. A threshold effect was observed at 35 μM, and maximal effect at 1.1 mM Mn²⁺. Ca²⁺ and calmodulin (CaM) alone did not facilitate p98 phosphorylation, but the presence of Mg²⁺ (10 mM) caused faint non-specific phosphorylation of p98. Ca²⁺ (0.03 to 3 mM) and CaM (1 to 10 μg/ml) significantly enhanced, whereas trifluoperazine (TFP) and Mg²⁺ inhibited Mn²⁺-stimulated p98 phosphorylation. Under the above incubation conditions, Mn²⁺-stimulated phosphorylation of p98 was also observed in isolated pancreatic acini, but not in cytosols from liver or kidney. 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. Accordingly, CaM PK III was partially purified from the rat pancreas using Mn²⁺-stimulated phosphorylation of pure EF-2 to assay its activity. Gel filtration chromatography indicated that the CaM-PK III had a Mᵣ of 150 kDa. Fractionation on the basis of isoelectric point, indicated that the majority of CaM-PK III activity was associated with a protein(s) of pI 4.5. A lesser amount of CaM-PK-III-like activity migrated to a pH of 8.4. These studies indicate that Mn²⁺ modulates CaM-PK III dependent phosphorylation of EF-2 in the exocrine pancreas and raise the possibility that Mn²⁺ can modulate pancreatic acinar cell function.