N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo

Abstract

There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.

cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines

“…In contrast, in S49 cells we did not detect any activation of this pathway by cCMP-AM or db-cCMP. db-cCMP is cleaved by esterases and the main metabolite 4-MB-cCMP arises, which is different from cCMP [17].…”

cCMP and cUMP occur in vivo

“…In agreement with this notion, Capan1 pancreas tumor cells contain cCMP as well [7]. To address this question, the effects of membrane-permeable cCMP analogs on endocrine and exocrine pancreas function could be studied [8,27]. …”

Interaction of cCMP with the cGK, cAK and MAPK Kinases in Murine Tissues

“…As previously discussed [ 9 ], cCMP/cAKRI regulates a large variety of cellular processes. However, its role in vivo might be less important than that recently described in an investigation into CREB-phosphorylation in S49 lymphoma cells [ 31 ]. MAPK stimulation of cAK via Ras/Raf has been demonstrated [ 32 ].…”