Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils

Two-dimensional electrophoretic analysis was used to assess quantitative an d qualitative changes in the expression and tyrosine phosphorylation of cyt oplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytopl asmic proteins appeared approximately constant during the pre-commitment pe riod, i.e., 6-24 h after induction of differentiation by 700 nM all-trans r etinoic acid. At the time of granulocytic phenotype formation (48-120 h), t he total level of tyrosine phosphorylation of cytoplasmic proteins increase d significantly. Tyrosine phosphorylation of cytoplasmic proteins in mature d blood neutrophils was significantly lower than that of cytoplasmic protei ns of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblottin g with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2b(k) antibodies sh owed that Erk2 was expressed and tyrosine-phosphorylated at different level s in HL-60 proliferating cells and in cells at all stages of differentiatio n. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phen otype formation and is accompanied by increasing activity of Erk2. An incre asing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentia tion). The appearance at this time of differentiation of a new set of tyros ine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL- 60 cells mediated by etoposide) together with an increasing number of apopt otic cells in the differentiating population strongly suggests that these p roteins are associated with the apoptotic process.