Identification of the Raf-1 signaling pathway used by cAMP to inhibit p42/p44 MAPK in rat lacrimal gland acini: role in potentiation of protein secretion.
OBJECTIVE
The lacrimal gland is primarily responsible for the aqueous portion of the tear film. Simultaneous addition of cholinergic agonists or growth factors with cAMP-dependent agonists potentiates secretion. Recent investigations revealed that cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity stimulated by cholinergic agonists and growth factors that could account for this potentiation. In this study the authors identify the signal transduction pathway used by cAMP to inhibit MAPK activity.


METHODS
Rat lacrimal gland acini were incubated with H89, an inhibitor of protein kinase A, before the addition of dibutyryl cAMP (dbcAMP, 10(-3) M) for 30 minutes. Basal MAPK and CREB activity and MAPK activity after stimulation with the cholinergic agonist carbachol (Cch) or epidermal growth factor (EGF) for 5 minutes was determined. The effect of dbcAMP on EGF receptor activity and basal and stimulated Ras, Raf-1, mitogen-activated protein kinase kinase (MEK), and MAPK activity was determined. The effect of a Rap-1 inhibitor, GGTI-298, on MAPK activity after the addition of dbcAMP was also determined.


RESULTS
H89 relieved the inhibition of cAMP on MAPK activity and inhibited CREB activity. Incubation with dbcAMP did not have any effect either on the EGF receptor or on Ras but significantly inhibited both basal and Raf-1 and MEK activity stimulated with Cch or EGF. GGTI-298 did not have any effect on cAMP-dependent decrease in MAPK activity.


CONCLUSIONS
The authors conclude that cAMP mediates the inhibition of MAPK by PKA in a Raf-1-dependent manner.
Funaki
Chika
C
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Hodges
Robin R
RR
Dartt
Darlene A
DA
D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007765:Lacrimal Apparatus / Q000187:drug effects* / Q000201:enzymology; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000494:pharmacology
D017362:Cyclic AMP Response Element-Binding Protein; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D003563:Cyclic CMP; D004815:Epidermal Growth Factor; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D002217:Carbachol; D000242:Cyclic AMP; D066246:ErbB Receptors; D019908:Proto-Oncogene Proteins c-raf; C546782:Raf1 protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide