Effect of retinoic acid on the phosphorylation of endogenous proteins in HL-60 cells

HL-60 cells were incubated with [32P]-Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two-dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic-acid-differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL-60 cells with [35S]-methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL-60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO-arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two-dimensional gels revealed that the phosphorylation of the 53-kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic-acid-induced differentiation process.