Altered protein kinase C in a mast cell variant defective in exocytosis.
The murine mast cell line PB-3c is dependent on interleukin 3 (IL-3) with respect to survival and proliferation. These cells also require IL-3 to display antigen-mediated serotonin release, which is coupled to a transient increase of cytosolic free calcium ([Ca2+]i). The antigen-mediated exocytosis is inhibited by phorbol 12-tetradecanoate 13-acetate (PTA), an activator of phospholipid/Ca2+-sensitive protein kinase. In contrast, the malignant mast cell variant PB-1 is IL-3 independent with respect to proliferation but is unable to undergo antigen-mediated exocytosis. Yet this cell line exhibits basal levels of [Ca2+]i, serotonin content, and numbers of IgE receptors comparable to those of PB-3c cells. Subcellular distribution studies revealed that the specific activity of cytosolic protein kinase C of PB-1 cells was only 40% of that found in PB-3c cells. Furthermore, the PB-1 cells showed a significantly higher specific activity of membrane-bound protein kinase C than PB-3c cells. Scatchard plot analysis of [3H]-phorbol 12,13-dibutyrate binding to intact PB-1 cells demonstrated the presence of 20% high-affinity (Kd = 6 nM) and 80% low-affinity (Kd = 60 nM) phorbol ester receptors, whereas PB-3c cells displayed only the low-affinity phorbol ester binding. Immunological characterization of protein kinase C from both cell lines revealed the presence of a normal 77-kDa protein kinase C holoenzyme in both cell lines. In addition, a 72-kDa protein kinase C-related protein band was found mainly in the membrane fraction of the PB-1 variant. It is suggested that this altered and membrane-bound form of protein kinase C may be involved in the blockage of the antigen-mediated exocytosis of PB-1 cells.
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins; D002460:Cell Line; D005089:Exocytosis; D014644:Genetic Variation; D007377:Interleukin-3 / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics*; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D007377:Interleukin-3; D010703:Phorbol Esters; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D012701:Serotonin; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium