Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP‐dependent protein kinases

Abstract

A series CAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for ;the two types of CAMP-binding sites in mammalian protein kinase type I. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that CAMP is bound to both sites in a comparable manner : the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of CAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation.The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (RJ-adenosine 3', 5'-monophosphorothioate [R,-CAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the CAMPbinding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize: the activation by CAMP. Indeed (RJ-CAMPS was found to inhibit CAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type I1 (R,)-CAMPS showed antagonistic activity, while with a CAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed.Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et al. (1982) Eur. J. Biochem. 122, 95 -991. This was based on measurements with impure preparations of (R,)-CAMPS and the R, and S, isomers adenosine 3', 5'-monophosphodimethylamidate, cAMPN(CH,), . The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (S,)-cAMPN(CH,), activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both CAMP binding sites.

Prostacyclin reverses platelet stress fibre formation causing platelet aggregate instability

“…4a). To confirm that the actin cytoskeletal rearrangements induced by PGI 2 were mediated by PKA the established PKA inhibitors Rp-8cpt-cAMPs (500 μM) and KT5720 (10 μM) were employed 26, 27 . The presence of Rp-8cpt-cAMPs (500 μM) and KT5720 (10 μM) blocked the ability of PGI 2 to reverse stress fibres, induce actin nodule formation, and reduce platelet spreading (Fig.…”

Coordination between proteasome impairment and caspase activation leading to TAU pathology: neuroprotection by cAMP

“…Rp-cAMPS is a PKA inhibitor that acts as a competitive antagonist of the cyclic-nucleotide-binding domains on PKA. 23 …”

Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

“…All PK inhibitors were prepared as 10 mM stock solutions dissolved in DMSO and further diluted with DMEM to obtain final concentrations. H9c2 cells (80% confluent) were pre‐incubated for 30 min in medium with or without the PKC inhibitor Ro 31‐8220 (10 μM; Davis et al ., ), and the PKA inhibitors KT 5720 (5 μM; Kase et al ., ) and R p‐8‐Cl‐cAMPS (50 μM; de Wit et al ., ) for 5 min prior to treatment with 1 μM PMA or 10 μM forskolin.…”