Differentiation of HL-60 cells is associated with an increase in the 35-kDa protein lipocortin I.
Lipocortin I, a 35-kDa protein, has been detected in terminally differentiated monocytes and neutrophils. This calcium-phospholipid binding protein appears to be identical to a 35-kDa protein that can serve as a substrate for the EGF-receptor/tyrosine kinase. We have used the human myelocytic cell line HL-60 to explore whether differentiation of hematopoietic cells is associated with changes in the level of lipocortin I. We find that differentiation of HL-60 cells toward the macrophage lineage by the addition of phorbol esters or vitamin D3 or toward neutrophils with dibutyryl cyclic AMP or dimethyl sulfoxide is accompanied by an increase in the cellular content of lipocortin I. In comparison, treatment of HL-60 cells with bryostatin 1, a compound that activates protein kinase C but does not differentiate HL-60 cells, did not effect the level of 35 kDa protein. We have developed a radioimmunoassay to quantitate this protein by using a polyclonal antibody to a synthetic amino terminal peptide of the 35-kDa protein. This antibody recognizes purified pig lung 35-kDa protein as well as a single 35-kDa protein in HL-60 and A-431 cells as determined by Western blotting and immune precipitation. Differentiated HL-60 cells contain 2.6-fold the amount of 35-kDa protein found in undifferentiated HL-60 cells. Our findings that the addition of phorbol esters to HL-60 cells results in an increase in the mRNA for the 35-kDa protein and in an increase in the incorporation of 35S-methionine into the protein suggest that transcriptional activation or increased stability of the mRNA is responsible for the increased rate of synthesis and accumulation of lipocortin I during differentiation of these cells. In the absence of added divalent cations, we have determined that in differentiated HL-60 cells 79% of lipocortin I protein is located in the cytosol while 21% of the total cellular protein is bound to the particulate fraction. The 35-kDa protein can be removed from the particulate fraction by incubation with chelators or treatment with phospholipase A2 or phospholipase C. Addition of the calcium ionophore A23187 to intact differentiated HL-60 cells causes the 35-kDa protein to associate with the particulate fraction of the cell, suggesting that modulation of intracellular calcium levels may play a role in changing the intracellular location of this protein.
William
F
F
Department of Medicine, University of Alabama, Birmingham 35294.
Mroczkowski
B
B
Cohen
S
S
Kraft
A S
AS
D017302:Annexins; D000001:Calcimycin / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003600:Cytosol / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000032:analysis / Q000166:cytology* / Q000378:metabolism; D009504:Neutrophils / Q000032:analysis / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017302:Annexins; D006023:Glycoproteins; D012333:RNA, Messenger; D000001:Calcimycin; D013755:Tetradecanoylphorbol Acetate