cAMP Analog Mapping of Epac1 and cAMP Kinase

Abstract

Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAP-KII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N 6 -benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10 -100-fold. Phenylthio substituents in position 8, particularly with MeO-or Cl-in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT-and 2-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.

Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC)

“…Commonly used cAMP analogs, such as 8-Br-cAMP, activate EPAC and PKA equally as well as does cAMP (24,25). However, analogs modified in the 6Ј position of the ribose (6-MB-cAMP) are poor EPAC activators and full PKA activators as compared with cAMP.…”

“…For that purpose, we took advantage of the availability of cAMP analogs that preferentially activate either pathway (24,25). OK cells, which endogenously express both transporters, and mouse kidney slices were treated with different cAMP analogs.…”

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

“…Nonetheless, we found that perfusion of hearts with the mixture of the specific PKA activator 6‐Bnz and the Epac activator CPT resulted in relatively very high haemodynamic functional recovery (Figure 7A and Table 2), significantly reduced LDH release and infarct size during reperfusion (Figure 7B–C). Similarly, 8‐Br, which simultaneously activates PKA and Epac (Christensen et al, 2003), markedly improved the recovery of haemodynamic function (Table 1, Figure 4) and prevented I/R injury (Figures 5 and 6) implying that both PKA and Epac are involved during cAMP‐induced cardioprotection. This conclusion is also supported by the fact that both PKA inhibitors (H‐89 and PKI) and the Epac inhibitor (ESI‐09), which appeared to have no effect on haemodynamic function recovery or I/R injury by themselves [see (Khaliulin et al, 2010), Table 1 and Figures 4, 5, 6], attenuated the cardioprotective effect of 8‐Br.…”

“…To investigate the involvement of PKC in mediating the effects associated with PKA and Epac activation by 8‐Br (Christensen et al, 2003), hearts were randomly divided into a control group, a 8‐Br group and a 8‐Br + chelerythrine group (five hearts per group, Figure 1). In the control group, the hearts were perfused for 40 min with no intervention prior to ischaemia.…”

Protein serine/threonine kinases (E.C. 2.7.1.-)

“…Endogenous activator cAMP PDK1 (Alessi et al, 1997) cGMP Rho Selective activators N 6 -Benzyl-cAMP (Christensen et al, 2003) ---…”