Induction of immediate early genes by Ca2+ influx requires cAMP-dependent protein kinase in PC12 cells.
Agents that activate cAMP-dependent protein kinase (PKA) as well as agents that increase intracellular calcium induce the expression of certain immediate early genes (IEGs). Recently, it has been demonstrated that the same cis-acting element in the 5' region of the c-fos gene has the ability to mediate both cAMP- and calcium-induced c-fos expression in PC12 cells (Sheng, M., McFadden, G., and Greenberg, M. (1990) Neuron 4, 571-582). Here we demonstrate that both cAMP- and calcium-mediated induction of c-fos and egr1 are dependent on PKA activity. Addition of either depolarizing concentrations of KCl or the calcium ionophore, ionomycin, to PC12 cells increased the expression of both c-fos and egr1, but these inductions were dramatically reduced in three PKA-deficient cell lines, 123.7, AB.11, and A126-1B2. Furthermore, pretreatment of PC12 cells with 20 microM H89, a specific inhibitor of PKA, inhibited forskolin, dibutyryl cAMP, and KCl-induced c-fos and egr1 induction, while having no effect on NGF induction. Likewise, in the PKA-deficient cells, NGF or an activator of protein kinase C induced c-fos and egr1 normally. To determine if PKA deficiency modifies the ability of Ca2+ to activate calcium-dependent kinases, autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in response to Ca2+ influx was determined. In parental PC12 cells, PC12 cells pretreated with H89, and PKA-deficient cell lines, CaM kinase was activated equivalently in response to KCl depolarization. These results suggest that PKA is not required for Ca(2+)-induced increase in CaM kinase activity and that the induction of IEGs in response to Ca2+ influx is PKA-dependent. Thus, the requirement for PKA resides at a point distal to the activation of calmodulin-dependent processes.
Ginty
D D
DD
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Glowacka
D
D
Bader
D S
DS
Hidaka
H
H
Wagner
J A
JA
D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D014157:Transcription Factors; D015759:Ionomycin; D011189:Potassium Chloride; D011494:Protein Kinases; D002118:Calcium