Absence of protein kinase C in nuclei of EL4 mouse thymoma cells.
Since evidence indicates that phorbol ester-induced production of interleukin 2 requires transcription, we investigated the possibility that the phorbol ester receptor acts directly in the nuclei of EL4 thymoma cells. Using a procedure that minimized plasma membrane contamination (as measured by 5'-nucleotidase activity) and maintained the integrity of the double nuclear membrane, we were unable to detect specific binding of [3H]phorbol 12,13-dibutyrate in nuclei of unstimulated cells. Treatment of cells with phorbol 12,13-dibutyrate (100 nM, 37 degrees C) for up to 6 h did not cause appearance of phorbol ester binding capacity in nuclei (4 +/- 8% of homogenate value; 5'-nucleotidase activity = 10 +/- 3%) despite translocation of 40% of the cytosolic binding capacity to the plasma membrane fraction. The failure to detect nuclear binding capacity in treated cells was not due to occupation of nuclear sites with unlabeled ligand; effective exchange binding was demonstrated by recovery of total homogenate binding capacity in treated cells of 82 +/- 13% of that in untreated cells. Treatment of isolated nuclei with DNase to liberate DNA binding proteins also failed to reveal any nuclear phorbol ester binding capacity. Assay of nuclei for protein kinase C enzymatic activity gave similar negative results. These data argue strongly against a direct action of the intact phorbol ester receptor (or the phorbol ester binding fragment) in the transcriptional activation of interleukin 2 in EL4 cells but cannot rule out the possibility of a role for the catalytic fragment.
D015720:5'-Nucleotidase; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000201:enzymology; D003851:Deoxyribonucleases / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D009708:Nucleotidases / Q000032:analysis; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000032:analysis*; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000378:metabolism; D013945:Thymoma / Q000201:enzymology* / Q000648:ultrastructure; D013953:Thymus Neoplasms / Q000201:enzymology* / Q000648:ultrastructure
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010703:Phorbol Esters; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D003851:Deoxyribonucleases; D009708:Nucleotidases; D015720:5'-Nucleotidase