Expression of c-fos and c-jun proteins and AP-1 binding activity during cell cycle progression of HL60 cells and phytohemagglutinin-stimulated lymphocytes.
The protein products of the c-fos (p62c-fos) and c-jun (p39c-jun) genes are members of the AP-1 transcription factor family and are thought to play important roles in the regulation of gene expression during the cell cycle. Most studies on the expression of these proteins in relation to the cell cycle have been performed at the mRNA level, and therefore do not give direct information about the presence of the proteins during the cell cycle. We have used Western blotting to investigate the presence of these proteins during the cell cycles of two different cellular systems: a continuously growing myeloid leukemic cell line, HL60, and normal cells stimulated into cycle, phyto- hemagglutinin (PHA)-stimulated normal human peripheral blood lymphocytes (PBL). The binding activity of transcription factor AP-1, which consists of dimers of Fos and Jun family proteins, was also studied using a gel shift assay. We found nuclear p62c-fos, p39c-jun, and AP-1 binding activity throughout the cell cycle both in HL60 cells and in PHA-stimulated PBL, and we postulate that these proteins are required throughout the cell cycle and not transiently in the G0 to G1 transition as previous mRNA studies have indicated. We demonstrated an uncoupling of AP-1 binding activity from p62c-fos, and p39c-jun AP-1 activity was expressed more strongly in the G1- and G2/M-phase enriched samples than in the S-phase enriched samples of HL60 cells, while levels of nuclear p62c-fos and p39c-jun were constant. Nuclei of unstimulated PBL from different donors expressed p62c-fos and p39c-jun, but AP-1 was not detected in the majority of samples. Following PHA stimulation of PBL, the increase in AP-1 activity was delayed with respect to the augmentation of p39c-jun expression. We also observed that cytoplasmic p62c-fos and p39c-jun were present in HL60 cells and PHA-stimulated PBL. However, no cytoplasmic p62c-fos was detected in unstimulated PBL, although in some cases cytoplasmic p39c-jun was detected, suggesting that subcellular compartmentalization of these proteins may occur under certain circumstances.
Tay
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DL
Department of Hematology, Royal Free Hospital School of Medicine, London, UK.
Hoffbrand
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AV
Wickremasinghe
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GR
D001483:Base Sequence; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D018922:HL-60 Cells / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010835:Phytohemagglutinins / Q000494:pharmacology*; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis* / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis* / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D009363:Neoplasm Proteins; D010835:Phytohemagglutinins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1