RIalpha Subunit Of Protein Kinase Research Articles

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S(p))-adenosine cyclic 3':5'-monophosphorothioate ((S(p))-cAMPS)) and the other a diastereoisomeric antagonist ((R(p))-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91-244)·C-subunit, by using fluorescence spectroscopy and amide H/(2)H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91-244), and the effects of cAMP, (S(p))-cAMPS, and (R(p))-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R(p))-cAMPS on amide exchange of the RIα(91-244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91-244). Surprisingly, (R(p))-cAMPS not only increased the affinity of RIα(91-244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R(p))-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R(p))-cAMPS-induced increase in affinity of RIα(91-244) toward the C-subunit indicates that (R(p))-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.

8-Cl-Adenosine Inhibits Proliferation and Causes Apoptosis in B-Lymphocytes via Protein Kinase A-Dependent and Independent Effects: Implications for Treatment of Carney Complex-Associated Tumors

Carney complex, a multiple neoplasia syndrome, characterized primarily by spotty skin pigmentation and a variety of endocrine and other tumors, is caused by mutations in PRKAR1A, the gene that codes for the RIalpha subunit of protein kinase A (PKA). PKA controls cell proliferation in many cell types. The cAMP analogue 8-Cl-adenosine (8-Cl-ADO) is thought to inhibit cancer cell proliferation. The objective of the study was to study the antiproliferative effects of 8-Cl-ADO on growth and proliferation in B-lymphocytes of Carney complex patients that have PKA defects and to determine whether 8-CL-ADO could be used as a therapeutic agent in the treatment of Carney complex-associated tumors. We used a multiparametric approach (i.e. growth and proliferation assays, PKA, and PKA subunit assays, cAMP and (3)H-cAMP binding assays, and apoptosis assays) to understand the growth and proliferative effects of 8-Cl-ADO on human B-lymphocytes. 8-Cl-ADO inhibited proliferation, mainly through its intracellular transport and metabolism, which induced apoptosis. PKA activity, cAMP levels, and (3)H-cAMP binding were increased or decreased, respectively, by 8-Cl-ADO, whereas PKA subunit levels were differentially affected. 8-Cl-ADO also inhibited proliferation induced by G protein-coupled receptors for isoproterenol and adenosine, as well as proliferation induced by tyrosine kinase receptors. 8-Cl-ADO in addition to unambiguously inhibiting proliferation and inducing apoptosis in a PKA-independent manner also has PKA-dependent effects that are unmasked by a mutant PRKAR1A. Thus, 8-Cl-ADO could serve as a therapeutic agent in patients with Carney complex-related tumors.

GEM 231, a second-generation antisense agent complementary to protein kinase A RIα subunit, potentiates antitumor activity of irinotecan in human colon, pancreas, prostate and lung cancer xenografts

GEM 231, a second-generation antisense oligonucleotide targeted against the RIalpha subunit of protein kinase A (PKA) was co-administered with the chemotherapeutic agent irinotecan, a topoisomerase-I inhibitor, to study the antitumor efficacy of the combination in nude mice bearing various human tumor xenografts. The combination treatment of GEM 231 and irinotecan produced enhanced and prolonged tumor-growth inhibition, compared with irinotecan monotherapy, against human colon (HCT-116), pancreas (Panc-1), prostate (PC3) and lung (SKMES) tumors in mice. The extent of tumor-growth inhibition, however, varied among the different tumor models studied. The tumor-growth inhibition depended on the dose of GEM 231 co-administered with irinotecan. The combination of GEM 231 (20 mg/kg, i.p., 5 days on 2 days off x 7) and irinotecan (50 mg/kg, i.v., qwk x 3) produced significantly longer tumor-growth delay than did irinotecan administered alone. Importantly, the co-administration of irinotecan and GEM 231 did not result in higher toxicity compared with monotherapies in the several tumor models tested. These results suggest that the use of irinotecan in combination with GEM 231 may increase the therapeutic index of irinotecan in cancer patients.

Mechanisms of FOXC2- and FOXD1-mediated Regulation of the RIα Subunit of cAMP-dependent Protein Kinase Include Release of Transcriptional Repression and Activation by Protein Kinase Bα and cAMP

We have reported recently that mice overexpressing the forkhead/winged helix transcription factor FOXC2 are lean and show increased responsiveness to insulin due to sensitization of the beta-adrenergic cAMP-PKA(+) pathway and increased levels of the RI alpha subunit of cAMP-dependent protein kinase (PKA) (Cederberg, A., Grønning, L. M., Ahren, B., Taskén, K., Carlsson, P., and Enerbäck, S. (2001) Cell 106, 563-573). In this present study, we reveal that FOXC2 and a related factor, FOXD1, specifically activate the 1b promoter of the RI alpha gene in adipocytes and testicular Sertoli cells, respectively. By deletional mapping, we discovered two different mechanisms by which the Fox proteins activated expression from the RI alpha 1b promoter. In 3T3-L1 adipocytes, an upstream region represses promoter activity under basal conditions. Bandshift experiments indicate that overexpression of FOXC2 promotes the release of a potential repressor from this region. In Sertoli cells, sequences downstream of the transcription start sites mediate the activating effect of FOXD1, and protein kinase B alpha/Akt1 strongly induces this effect. Furthermore, we show that an inactive FOXD1 mutant lowers the cAMP-mediated induction of the RI alpha 1b reporter construct. In summary, winged helix transcription factors of the FOXC/FOXD families function as regulators of the RI alpha subunit of PKA and may integrate hormonal signals acting through protein kinase B and cAMP in a cell-specific manner.

Caffeine-induced telencephalic vesicle evagination in early post-implantation mouse embryos involves cAMP-dependent protein kinase (PKA) inhibition.

Other studies have shown that caffeine accelerates telencephalic vesicle evagination in early post-implantation mouse embryos. The present study examines the effect of caffeine on gene modulation in post-implantation mouse embryos. Using mRNA differential display, we observed that caffeine increased gene expression of the regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA). RT--PCR analysis confirmed an increase in expression of this gene in caffeine-exposed embryos when compared with saline-treated controls. Using a fluorescent substrate of PKA, we found that PKA activity in the presence of cAMP was lower in caffeine-treated embryos than in controls. Treatment with H89 and PKI(12-24)amide, two inhibitors of PKA activity, mimicked the effects of caffeine on telencephalic vesicle formation. Together these data suggest that in early post-implantation mouse embryos caffeine modulates gene expression of the RI alpha subunit of PKA and that caffeine-induced inhibition of PKA activity plays a role in early telencephalic evagination.

Biodistribution and metabolism of a mixed backbone oligonucleotide (GEM 231) following single and multiple dose administration in mice.

Biodistribution and metabolism of a mixed backbone oligonucleotide (MBO), GEM 231, targeted to the RIalpha subunit of protein kinase A has been studied in normal and tumor xenografted mice. The study has been carried out using [35S]-labeled MBO following single and multiple administrations of doses varying from 2 to 50 mg/kg. MBO showed wide tissue distribution following intravenous and subcutaneous administration. The highest concentration of MBO was in the kidney and liver. The general disposition of MBO was followed by digitized autoradiographic pictures of tumored mice and further confirmed wide tissue disposition and also showed defined intratumor uptake of MBO. Multiple dose administration showed increased disposition in the majority of the tissues/organs, with the exception of the kidneys. Analysis of the extracted MBO by polyacrylamide gel electrophoresis (PAGE) showed the presence of primarily intact MBO along with its degraded forms. Based on our radioactivity levels, the primary route of excretion was in urine, analysis of which showed mainly degraded forms of MBO.