Opposing regulatory effects of protein kinase C on the cAMP cascade in human HL-60 promyelocytic leukemia cells.
The functional role of protein kinase C in the cAMP signaling cascade was investigated in human promyelocytic leukemia (HL-60) cells. Protein kinase C activation after short exposure to 100 nM phorbol 12-myristate 13-acetate (PMA) increased the intracellular cAMP level up to 3- to 5-fold after 30 min. Such enhancement was almost completely blocked by the selective protein kinase C inhibitor bisindolylmaleimide (GF 109203X). In addition, PMA, but not 4-alpha-PMA, synergistically elevated cAMP levels when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) treatment in digitonin-permeabilized cells. The results indicate that protein kinase C directly increases adenylyl cyclase activity and synergistically enhances it, when it is simultaneously activated otherwise. On the other hand, a 10-min treatment with PMA cut the cAMP accumulation induced by histamine, prostaglandin E2, or isoproterenol by 50-70%. However, the binding affinity and total binding of [3H]histamine to membrane receptors was not effected by PMA, suggesting that the site of protein kinase C's action is not at the receptor level. Western blot analysis of protein kinase C isozymes revealed that PMA (100 nM) caused translocation of cytosolic protein kinase C such as alpha, beta and epsilon to the particulate/membrane fraction. Treatment with a lower concentration of PMA (10 nM) translocated the protein kinase C-epsilon within 2 min, while it had little effect on the translocation of protein kinase C-alpha and -beta up to 20 min. However, simultaneous treatment with 10 nM PMA plus histamine for 5 min significantly inhibited the histamine-mediated cAMP generation, indicating that the protein kinase C-epsilon could be involved in the inhibition of receptor-mediated cAMP generation. Taken together, we conclude that PMA, through the activation of protein kinase C, has two opposite effects on the cAMP signaling cascade in HL-60 cells: a direct activation of adenylyl cyclase and an inhibition of receptor-mediated signal transduction.
Byung-Chang
S
S
Department of Life Science, Pohang University of Science and Technology, South Korea.
Se-Young
C
C
Jang-Soo
C
C
Kyong-Tai
K
K
D000262:Adenylyl Cyclases / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D018922:HL-60 Cells; D006632:Histamine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D011968:Receptors, Histamine / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D011968:Receptors, Histamine; D006632:Histamine; D000242:Cyclic AMP; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate