Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi-endosomal fraction.
The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.
Geoffroy
V
V
INSERM U30, Groupe Hospitalier Necker-Enfants Malades, Paris, France.
Fouque
F
F
Nivet
V
V
Clot
J P
JP
Lugnier
C
C
Desbuquois
B
B
Benelli
C
C
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000494:pharmacology*; D011992:Endosomes / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D006056:Golgi Apparatus / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D009281:Naphthalenes / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D015363:Quinolones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D004791:Enzyme Inhibitors; D009281:Naphthalenes; D010726:Phosphodiesterase Inhibitors; D015363:Quinolones; C061465:calphostin complex; C021294:cilostamide; C010192:9-(2-hydroxy-3-nonyl)adenine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D000225:Adenine; D013755:Tetradecanoylphorbol Acetate