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

Abstract

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.

Alterations in Protein Expression in HL‐60 Cells during Etoposide‐Induced Apoptosis Modulated by the Caspase Inhibitor ZVAD.fmk

“…Finally, we screened cytosolic and nuclear protein maps in HL‐60 control cells, cells induced to apoptosis, or cells treated with a broad caspase inhibitor. In our previous studies,18 we demonstrated that the synthesis of new proteins and protein modification occur during the induction of HL‐60 cells to granulocytic differentiation. We suggested that changes in protein synthesis and protein tyrosine phosphorylation seen in differentiating HL‐60 cells reflect both the formation of the differentiated granulocyte phenotype and the apoptotic process.…”

Translocation of transcription regulators into the nucleus during granulocyte commitment of HL-60 cells

“…We also found tyrosine-phosphorylated proteins that were specific to the nucleus of differentiated HL-60 cells; some of these proteins were tyrosine phosphorylated in the cytoplasm before being moved into the nucleus and others were tyrosine phosphorylated after nuclear translocation. We have previously shown (Treigyte et al 2000) that tyrosine phosphorylation of cytosolic proteins increased significantly in HL-60 cells undergoing differentiation. The patterns of nuclear proteins revealed by 2-DE showed that soon after differentiation was induced, the tyrosine phosphorylation level of some proteins increased markedly, especially with acidic and neutral pI, whereas some tyrosine-phosphorylated proteins had undergone dephosphorylation either markedly or completely.…”

The effect of inhibiting topoisomerase I and II on the antiapoptotic response associated with pro-inflammatory crystals of calcium pyrophosphate dihydrate in human neutrophils

“…Camptothecin has been shown to induce DNA strand breaks in vivo, and is related to the formation of a camptothecin-induced cleavable topoisomerase-DNA complex [37]. Another factor common to both camptothecin and topoisomerase II inhibitors is that the cytotoxic effects induced by these drugs have been shown to correlate to the degree of tyrosine phosphorylation of cellular proteins other than the topoisomerases themselves [38,48]. Interestingly, we and others have demonstrated previously that neutrophils undergo significant tyrosine phosphorylation when stimulated with fMLP or CPPD crystals [23,49,50].…”