Opposing regulatory effects of protein kinase C on the cAMP cascade in human HL-60 promyelocytic leukemia cells

Abstract

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-α-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) (GTPγS) 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 E 2, or isoproterenol by 50–70%. However, the binding affinity and total binding of [ 3 H ]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 α, β and ε to the particulate/membrane fraction. Treatment with a lower concentration of PMA (10 nM) translocated the protein kinase C-ε within 2 min, while it had little effect on the translocation of protein kinase C-α and -β 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-ε 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.