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Effect of retinoic acid on the phosphorylation of endogenous proteins in HL‐60 cells
Author(s) -
Spearman Terry N.,
Fontana Joseph A.,
Butcher Fred R.,
Durham John P.
Publication year - 1989
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041380218
Subject(s) - retinoic acid , phosphorylation , endogeny , cellular differentiation , microbiology and biotechnology , biology , tretinoin , in vitro , protein phosphorylation , biochemistry , methionine , cell , gel electrophoresis , chemistry , amino acid , protein kinase a , gene
HL‐60 cells were incubated with [ 32 P]‐P i 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 [ 35 S]‐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.