Subunit Of cAMP-dependent Kinase Research Articles

Tamoxifen enhances the anti-cancer effect of cantharidin and Norcantharidin in pancreatic cancer cell lines through inhibition of PKC pathway

Cantharidin (1) as well as norcantharidin or endothall (2) have been described to be inhibitors of protein phosphatase 2A, but were reported to be inactive as inhibitors of calcineurin. Using an HPLC-assay, we have discovered that 1 and 2 are inhibitors of calcineurin-dependent (appKi = 10.8 and 3.26 μM, respectively) dephosphorylation of a phosphopeptide derived from the RII subunit of c-AMP dependent kinase, H-DLDVPIPGRFDRRVS(PO4)VAAE-OH (7), which is commonly used in calcineurin assays. The phosphatase inhibitor 6 also inhibits the production of IL-2 in Jurkat cells, a calcineurin dependent process, with IC50 = 4.7 μM, indicating that some of the biological activity of this substance class may be due to calcineurin inhibition, in addition to the established PP2A inhibition.

Rapamycin decreases tau phosphorylation at Ser214 through regulation of cAMP-dependent kinase

Preventing or reducing tau hyperphosphorylation is considered to be a therapeutic strategy in the treatment of Alzheimer’s disease (AD). Rapamycin may be a potential therapeutic agent for AD, because the rapamycin-induced autophagy may enhance the clearance of the hyperphosphorylated tau. However, recent rodent studies show that the protective effect of rapamycin may not be limited in the autophagic clearance of the hyperphosphorylated tau. Because some tau-related kinases are targets of the mammalian target of rapamycin (mTOR), we assume that rapamycin may regulate tau phosphorylation by regulating these kinases. Our results showed that in human neuroblastoma SH-SY5Y cells, treatment with rapamycin induced phosphorylation of the type IIα regulatory (RIIα) subunit of cAMP-dependent kinase (PKA). Rapamycin also induced nuclear translocation of the catalytic subunits (Cat) of PKA and decreases in tau phosphorylation at Ser214 (pS214). The above effects of rapamycin were prevented by pretreatment with the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126. In addition, these effects of rapamycin might not depend on the level of tau expression, because similar results were obtained in both the non-tau-expressing wild type human embryonic kidney 293 (HEK293) cells and HEK293 cells stably transfected with the longest isoform of recombinant human tau (tau441; HEK293/tau441). These findings suggest that rapamycin decreases pS214 via regulation of PKA. Because tau phosphorylation at Ser214 may prime tau for further phosphorylation by other kinases, our findings provide a novel possible mechanism by which rapamycin reduces or prevents tau hyperphosphorylation.

Protein kinase A regulatory subunit distribution in medulloblastoma

BackgroundPrevious studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors.MethodsThe distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding.ResultsR2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma.ConclusionsA different distribution of cAMP dependent protein kinases has been observed in medulloblastoma.

Aggregates of cAMP-dependent kinase RIα characterize a type of cholinergic neurons in the rat brain

Acetylcholine is synthesized by different types of neurons, showing a distinct biochemical phenotype. Aggregates of RIα regulatory subunit of cAMP-dependent protein kinases are visualized by immunohistochemistry only in some cholinergic neurons, since they tightly colocalize with two different markers, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). These neurons are present mainly in brain areas related to the limbic system. None of the other regulatory subunits of cAMP dependent kinases colocalize with cholinergic markers.

7-Oxa[2.2.1]bicycloheptane-2,3-dicarboxylic acid derivatives as phosphatase inhibitors

Abstract Cantharidin (1) as well as norcantharidin or endothall (2) have been described to be inhibitors of protein phosphatase 2A, but were reported to be inactive as inhibitors of calcineurin. Using an HPLC-assay, we have discovered that 1 and 2 are inhibitors of calcineurin-dependent (appKi = 10.8 and 3.26 μM, respectively) dephosphorylation of a phosphopeptide derived from the RII subunit of c-AMP dependent kinase, H-DLDVPIPGRFDRRVS(PO4)VAAE-OH (7), which is commonly used in calcineurin assays. The phosphatase inhibitor 6 also inhibits the production of IL-2 in Jurkat cells, a calcineurin dependent process, with IC50 = 4.7 μM, indicating that some of the biological activity of this substance class may be due to calcineurin inhibition, in addition to the established PP2A inhibition.

Binding of two fluorescent cAMP analogues to type I and II regulatory subunits of cAMP-dependent protein kinases

Binding of two long wavelength fluorescent cAMP analogues, 8-thioacetamido-fluorescein-cAMP (SAF-cAMP) and 8-thioacetamido-rhodamine-cAMP (SAR-cAMP), to the RI (from bovine muscle) and RII (from bovine heart) regulatory subunits of cAMP dependent kinases has been studied. Displacement of [ 3H]cAMP from RI and RII and equilibrium dialysis measurements show that the fluorescent nucleotides are high affinity ligands for the cAMP binding sites. The binding is characterized by complex fluorescence spectral and fluorescence anisotropy changes, more evident for the fluorescein than for the rhodamine derivative. The fluorescence excitation spectrum of the bound SAF-cAMP is characterized by the appearance of a red shifted shoulder at 500–510 nm excitation wavelength region. Any change of the bound/free ratio in a solution equilibrium is accompanied by changes in fluorescence and anisotropy signals which are best detected at suitable wavelengths. It is proposed that fluorescence and anisotropy changes can distinguish between binding to type B (slow dissociating) and A (fast dissociating) cAMP binding sites of regulatory subunits. Applications of the fluorescent nucleotides to kinase localization and cAMP determination in living cells are discussed.

Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II.

The antigenic regions of the type II regulatory subunit of cAMP-dependent kinase from bovine heart have been correlated with the previously established domain structure of the molecule. Immunoblotting with both serum and monoclonal antibodies of fragments generated by limited proteolysis or chemical cleavage of the R-subunit established that the major antigenic sites were confined to the amino-terminal portion of the polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested that one or more of the high affinity serum antibody recognition sites were further restricted to residues 91-145. This amino-terminal portion of the R-subunit includes the hinge region which is particularly sensitive to proteolysis, allowing the R-subunit to be cleaved readily into a COOH-terminal domain which retains the cAMP-binding sites and an NH2-terminal fragment which appears to be the major site for interaction of the R-subunits in the native dimer. Monoclonal antibodies that recognized determinants on both sides of this hinge region were characterized and their specific recognition sites localized. Accessibility of antigenic sites in the holoenzyme in contrast to free R2 was compared. Although cAMP did tend to slightly increase the affinity of the holoenzyme for one of the monoclonal antibodies, all of the antigenic sites clearly were exposed and accessible in the holoenzyme. Furthermore, despite the presumed close proximity of antigenic sites to interaction sites between the R- and C-subunits, in no case did binding of antibody to the holoenzyme promote dissociation of the complex. The fact that the monoclonal antibodies would precipitate holoenzyme as well as free R2 was used to ascertain the importance of specific amino acid residues in the interaction of the R- and C-subunits. cAMP-binding domains were isolated following limited proteolysis with chymotrypsin and thermolysin. These fragments differed by only three amino acid residues at the NH2-terminal end. U of these fragments in conjunction with immunoadsorption established that the chymotryptic fragment, which contained the Asp-Arg-Arg preceding the site of autophosphorylation, was capable of forming a stable complex with the C-subunit. In contrast, the thermolytic fragment which differed by only those three residues no longer complexed with the C-subunit, indicating that the arginine residues not only contribute to the specificity of the phosphorylation site but also are an essential component for energetically stabilizing the holoenzyme complex.