Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase Cin 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. Inj ection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activat or 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 PK C alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbo l 12-myristate 13-acetate treatment caused a time-dependent increase in cAM P PDE activity in the GE fraction (96% at 30 min). Addition of the catalyti c 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 f rom 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the T riton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprec ipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulate d 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, whi ch were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak whic h was activated (threefold) by 5 mu M cGMP and inhibited (87%) by 25 mu M E HNA, 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 changi ng the K-m value. These results suggest that PKC selectively activates a PD E2, cGMP-stimulated isoform in the GE fraction.