Development of oxidase 'priming' in maturing HL60 cells: correlation with protein expression and tyrosine phosphorylation.
The mechanisms involved in 'priming' of the oxidase response of neutrophils are unknown. Two major problems are encountered in using circulating neutrophils; firstly, prior exposure to circulating 'priming' cytokines cannot be controlled and secondly, non-intentional 'priming' during cell separation can occur. In this study, these problems were avoided by differentiating the promyeloid leukaemic cell line, HL60, towards granulocytes using dibutyrl cyclic AMP, to produce a 'virgin cell' model system. We have demonstrated that the ability of substance P to both prime the oxidase response and induce tyrosine phosphorylation increased during differentiation. The major tyrosine-phosphorylated protein, with molecular weight of 74 kDa, was not recognised by anti-c-raf1 antibodies. Furthermore, c-raf1 expression rapidly declined during HL60 cell granulocytic differentiation. This data shows that although there was no simple relationship between c-raf quantity and priming, the data were consistent with tyrosine phosphorylation of a 74 kDa protein being important for oxidase 'priming'.
Lloyds
D
D
University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Hallett
M B
MB
D002454:Cell Differentiation; D002460:Cell Line; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D010088:Oxidoreductases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D018000:Receptors, Peptide / Q000378:metabolism; D015398:Signal Transduction; D013373:Substance P / Q000494:pharmacology
D011506:Proteins; D011518:Proto-Oncogene Proteins; D018000:Receptors, Peptide; D013373:Substance P; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf