Abstract
Phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol acetate (TPA) activate the calcium- and phospholipid-dependent protein kinase C and enhance three biological responses (prolactin release, prolactin synthesis, and cell stretching) in GH4C5 rat pituitary cells. We have examined several actions on GH4C5 cells of TPA and two other classes of protein kinase C activators, synthetic cell permeant dioleins and bryostatins isolated from the marine bryozoan Bugula neritina. Bryostatins 1 and 2 (B1 and B2, respectively) competed for [3H]phorbol 12,13-dibutyrate binding to the protein kinase C complex in intact cells nearly equipotently with TPA. B1 and B2, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoylglycerol (Di8) as well as TPA each activated partially purified protein kinase C from GH4C5 cells. B1, B2, and TPA each enhanced the acute release of prolactin from GH4C5 cells to a similar maximal extent. B1, B2, and TPA also enhanced prolactin synthesis. However, B1 and B2 were only partial agonists because they enhanced prolactin synthesis to a lesser maximal extent than did TPA and, given in combination, they reduced TPA-enhanced prolactin synthesis. OAG and Di8 stimulated prolactin release (to a lesser maximal extent than TPA) and did not stimulate prolactin synthesis. Pretreatment with OAG did not reduce TPA-stimulated prolactin release or synthesis. B2 and TPA induced cell stretching in GH4C5 cells, whereas B1, OAG, and Di8 induced little if any stretching. B1, but not B2, given in combination with TPA antagonized TPA-induced stretching but did not reduce thyrotropin-releasing hormone- or epidermal growth factor-induced stretching. We conclude that the bryostatins, phorbol esters, and dioleins bind to the same site on the protein kinase C complex to activate the enzyme, but they alter three biological responses in GH4C5 cells with selectivities and efficacies that differ. We propose that different activators of protein kinase C (such as bryostatins, dioleins, and phorbol esters) may elicit different cellular responses by altering the substrate specificity or activating multiple forms of the kinase.
Highlights
Phorbol ester tumor promoters such as 12-0-tetra- protein kinase C complex to activate the enzyme, but decanoylphorbol acetate (TPA) activate the calcium- they alter three biological responses in G H & s cells and phospholipid-dependent protein kinase C and en- with selectivities anedfficacies that differ
B1 and B2 were only partial agonists because they quent findings that the phorbol ester binding site copurifies enhanced prolactin synthesis to a lesser maximal ex- with protein kinase C (6, 7) and that certain phorbol ester tentthandid TPA and, given in combination, they tumor promoters can substitute in a competitive manner for reduced TPA-enhanced prolactin synthesis
Di8 stimulated prolactin release and dindot stimulate prolactin synthesis
Summary
1ml of lysis buffer (20 mM Tris, 10 mM EGTA, 2 mM EDTA, 10 pg/ ml leupeptin). The cells were lysed by passage through a 25 gauge needle eight times, and thesuspension was centrifuged a t 12,000 X g for 15 min at 4 "C. Cells were treated with increasing concentrations (10"o-10-6 M) of bryostatin 1, 2, TPA, or 10-100 pg/ml ofOAG or Di8 in 1ml of this medium for 30 min at 37 "C. For experiments with OAG and Di8, the cells were washed extensively, and bovine albumin was omitted from the incubation medium. This procedure reduced unstimulated levels of prolactin in the medium and maximized the response seen with the synthetic dioleins. To measure the Methods effects of synthetic dioleins on prolactin synthesis, triplicate dishes were washed threetimes with Eagle's minimal essential medium. Cells were washed with Cos-equilibrated F-10+ and in- prolactin accumulation in the medium over 24 h represents >90%. Bryostatin1and 2 enhanced therelease of prolactin in a dose-dependent manner
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