pp75: A novel tyrosine-phosphorylated protein that heralds differentiation of HL-60 cells.
The human promyeloid cell line HL-60 differentiates toward monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or dibutyryl cAMP, respectively. When nondifferentiated cells were incubated for 20 min with 2 mM H2O2 and 0.1 mM sodium orthovanadate to inhibit their protein-tyrosine-phosphatase activity (Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265, 2896-2902), we found marked tyrosine phosphorylation of a single major protein of 53 kDa. Induction of differentiation of HL-60 cells was accompanied by the appearance of an additional major cytosolic tyrosine-phosphorylated protein of 75 kDa (pp75). In dibutyryl cAMP-treated cells, tyrosine phosphorylation of pp75 peaked after 24 h and then declined rapidly. In 1,25(OH)2D3-treated cells, increased tyrosine phosphorylation was detected as early as 2 h and peaked after 3 days, whereas the presence of differentiated phenotypes, assessed by the capacity of the cells to reduce nitro blue tetrazolium, was detected no earlier than 24 h. Doses of 1,25(OH)2D3 as low as 1 nM induced the appearance of pp75 at a stage where almost no differentiation measured by nitro blue tetrazolium reduction was detected. Phosphorylation of pp75 was not stimulated by adriamycin, which induced growth arrest without initiation of differentiation. pp75 could also be detected in U-937, a monocytic cell line that is more advanced in its differentiation state, and also in terminally differentiated circulating human monocytes treated with H2O2/vanadate. pp75 underwent in vitro tyrosine phosphorylation in cytosolic extracts derived from 1,25(OH)2D3-induced HL-60 cells, but not in extracts derived from uninduced cells. Our results raise the possibility that tyrosine phosphorylation of pp75 may be a common early event that heralds the differentiation of HL-60 cells into both the monocytic and granulocytic pathways.
Bushkin
I
I
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Roth
J
J
Heffetz
D
D
Zick
Y
Y
D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation; D004317:Doxorubicin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D010750:Phosphoproteins; D014638:Vanadates; D014443:Tyrosine; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D002117:Calcitriol