Activation of α-Protein Kinase C Leads to Association with Detergent-Insoluble Components of GH4C1Cells

Abstract

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.