Manganese enhances phosphorylation of a 47 kD protein in retinoic acid-induced HL-60 cells.

We previously observed that HL-60 cells treated with manganese (Mn) during differentiation displayed an enhanced oxidative burst. Since a Mn-dependent kinase has been identified and phosphorylation is involved in burst activation, the objective of this research was to identify proteins in retinoic acid-induced HL-60 cells whose phosphorylation after phorbol myristate acetate (PMA) stimulation was affected by Mn treatment. Cells received Mn during differentiation and were then harvested, labeled with [32]P-orthophosphate, and stimulated with PMA. Cytosolic proteins were separated by isoelectric focusing, SDS-PAGE, and two-dimensional (2-D) gel electrophoresis. Time studies showed that Mn treatment did not alter the rate of PMA activated phosphorylation. Isoelectric focusing revealed that PMA stimulation resulted in the appearance of three phosphoproteins at pI's of 6.8, 7.3, and 7.8. Size separation gels showed a 200% increase in phosphorylation of a 47 kD protein in Mn-treated cells after stimulation. The 2-D gels showed that the pI of this protein was 6.8. Therefore, Mn treatment resulted in greater phosphorylation of a 47 kD protein, pI 6.8, in phorbol ester-stimulated cells.