Conditional activation of cAMP signal transduction by protein kinase C. The effect of phorbol esters on adenylyl cyclase in permeabilized and intact cells.

B.H Morimoto,D.E Koshland

doi:10.1016/s0021-9258(17)41743-1

B.H Morimoto, D.E Koshland

Open Access

https://doi.org/10.1016/s0021-9258(17)41743-1

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Abstract

To understand the convergence of cAMP and protein kinase C signal transduction, adenylyl cyclase isozyme identification and biochemical studies were performed on the HT4 neural cell line. In HT4 cells, basal cAMP production by adenylyl cyclase types I and VI were unaffected by phorbol esters, nor did phorbol esters have any effect on forskolin-induced cAMP production. However, phorbol esters synergistically increased cAMP production when adrenaline receptors were simultaneously activated, indicating a conditional activation of cAMP production by phorbol esters. A permeabilized cell preparation was used to analyze the mechanism by which phorbol esters were affecting cAMP production. This preparation enables G-proteins to be activated directly by GTP gamma S or bacterial toxins. In the permeabilized cell preparation, phorbol esters enhanced cAMP produced by GTP gamma S-activated G-protein. A stimulatory G-protein pathway may be involved since phorbol esters synergistically increased cAMP production by cholera toxin, yet had no effect on that produced by pertussis toxin. In this cell culture model, phorbol esters stimulate cAMP production only when some component of the cAMP cascade is simultaneously activated. Moreover, the pattern of modulation suggests that protein kinase C acts on an activated component of the second messenger system, such as the G-protein or the coupling of the G-protein with adenylyl cyclase, and not on the resting state of the protein components.

Highlights

To understand the convergence ofCAMP and protein ing the effect of phorbol esters on forskolin-stimulated type I kinase C signal transduction, adenylyl cyclase isozyme adenylyl cyclase activity in stably transfected cel(l3s) or tranidentification and biochemical studies were performedsiently transfected cells[2] exemplifies the complexity of these on the HT4 neural cell line
Affected by phorbol esters, nor did phorbolesters have Neural cell lines provide a homogeneous population of cells any effecton forskolin-inducedCAMPproduction
In one succhell ductiown heandrenalinreeceptorws erseimultaline, HT4, CAMP production is importantin the modulation of ntCCheAAAeoM Mum psPPeleprpycmrrhooaaeddcnauutbiisccvimtltaiiiozotenebnddy.b,Twcyiehnhlpilidschiphpocrarrpebtephiponaolagrrreabasatottieiloorcnensos.tnweednrasiastbiwoulenessraeedlGaa-tfpocfertcioavttnioaenfatigniloysnzctnaeheneeludlurtoelloamtrrnapgrnoe-ssrtmeaprloimntetsleipervvoeastneteeincostrsnieaott(fii6oCo)nnA.(oT5Mfh,n6ePe)cpu.oHerrorTrste4irlsaactnetesenlmlstseiwtletexeivtrpharsteietsohcsnreeibntioinotchCnr,AesMaahsnoPedrt-in to be activated directbly GTP+ or bacterial toxins.In levels appears to involve co-activation of CAMP and protein the permeabilized cell preparation, phorbol esters en- kinase C signal transduction pathways' and may result in a hanced C A M P produced by GTPyS-activated G-protein. "molecular switch," changing the cell from short- to long-term

Summary

EXPERIMENTAL PROCEDURES

System, such as the G-proteinor the coupling of theG- Chemicals and Reagent~--'~~I-cAMradPioimmunoassay(RIA) was protein with adenylyl cyclase, and not on the resting obtained from Amersham. 5'-(N-Ethyl)carboxyamidoadenosine state of the protein components. The effect of phorbol esters on forskolin-stimulated CAMP production was verified inthe digitonin-permeabilized cell preparation. The stimulatory a subunit of the heterotrimeric Gprotein (Gas) directly interacts and activatesadenylyl cyclase, whereas the inhibitory a subunit (Gai) inhibits adenylyl cyclase by an unknown mechanism [11].if the increase in CAMP production upon phorbol ester presentation is the result of G-protein activation or coupling between the G-protein and adenylyl cyclase, either stimulation of a Gas or inhibition of a. To assess which G-protein pathway wasinvolved in phorbol ester-induced synergism,bacterial toxins which covalently modify either of the G-proteins wereused.Cholera toxin ADP-ribosylates the Gas subunit, resulting in theinhibition of the intrinsicGTPase activity and stabilization of the activatedconformation

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DISCUSSION