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) du ring 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 pr oteins in retinoic acid-induced HL-60 cells whose phosphorylation afte r phorbol myristate acetate (PMA) stimulation was affected by Mn treat ment. Cells received Mn during differentiation and were then harvested , labeled with [32] P-orthophosphate, and stimulated with PMA. Cytosol ic proteins were separated by isoelectric focusing, SDS-PAGE, and two- dimensional (2-D) gel electrophoresis. Time studies showed that Mn tre atment did not alter the rate of PMA activated phosphorylation. Isoele ctric focusing revealed that PMA stimulation resulted in the appearanc e of three phosphoproteins at pi's of 6.8, 7.3, and 7.8. Size separati on gels showed a 200% increase in phosphorylation of a 47 kD protein i n 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 p hosphorylation of a 47 kD protein, pI 6.8, in phorbol ester-stimulated cells. (C) 1998 Wiley-Liss, Inc.