Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils.
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.
Treigyté
G
G
Laboratory of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
Navakauskiené
R
R
Kulyté
A
A
Gineitis
A
A
Magnusson
K E
KE
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005047:Etoposide / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007525:Isoelectric Focusing; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
D010750:Phosphoproteins; D019000:Phosphotyrosine; D014212:Tretinoin; D005047:Etoposide; D019950:Mitogen-Activated Protein Kinase 1