Activation of alpha-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells.
TRH and phorbol dibutyrate (PDBu) stimulate PRL secretion and synthesis from GH4C1 rat pituitary cells through activation of protein kinase C (PKC). TRH responses are mediated by increases in cellular levels of two PKC activators, Ca2+ and diacylglycerol (DAG), whereas PDBu acts as a DAG analog. We conducted experiments to compare the effects of Ca2+ and PDBu/DAG on alpha-PKC redistribution and to determine to what components of the particulate fraction activated alpha-PKC associates. Subcellular fractionation experiments demonstrated that TRH and PDBu both caused chelator-stable association of alpha-PKC with the particulate fraction. In contrast, Ca2+-mediated association with the particulate fraction was not chelator stable. Immunocytofluorescence experiments also demonstrated that TRH, PDBu, and increased cytosolic Ca2+ (due to ionomycin or K+ depolarization) caused redistribution. The effect of TRH was rapid and transient, similar to TRH stimulation of phospholipase C. The translocated alpha-PKC in the particulate fraction from TRH- or PDBu-treated cultures was not solubilized with Triton X-100. In comparable studies using an immunofluorescence assay, alpha-PKC immunofluorescence remained in detergent-insoluble preparations from TRH- and PDBu-stimulated, but not resting cells. The association of activated alpha-PKC with chelator- and detergent-insoluble material suggested that activated alpha-PKC may be associated with membrane and cytoskeletal components.
Kiley
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W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946.
Jaken
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D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D005989:Glycerides / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism; D012995:Solubility; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D005989:Glycerides; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D013973:Thyrotropin-Releasing Hormone; D011493:Protein Kinase C; D002118:Calcium