Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: Levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIα) was present in all the cells investigated, in contrast to the other R subunits (RIβ, RIIα, and RIIβ). Furthermore, three T cell lines and one B cell line expressed only RIα and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIα protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [³H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes. J. Cell. Physiol. 177:85–93, 1998. © 1998 Wiley-Liss, Inc.     

Protein kinase a alterations in endocrine tumors

Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis.     

Regulation of rat alveolar type 2 cell proliferation in vitro involves type II cAMP-dependent protein kinase

To elucidate the role of cAMP and different cAMP-dependent protein kinases (PKA; A-kinase) in lung cell proliferation, we investigated rat alveolar type 2 cell proliferation in relation to activation or inhibition of PKA and PKA regulatory subunits (RIIalpha and RIalpha). Both the number of proliferating type 2 cells and the level of different regulatory subunits varied during 7 days of culture. The cells exhibited a distinct peak of proliferation after 5 days of culture. This proliferation peak was preceded by a rise in RIIalpha protein level. In contrast, an inverse relationship between RIalpha and type 2 cell proliferation was noted. Activation of PKA increased type 2 cell proliferation if given at peak RIIalpha expression. Furthermore, PKA inhibitors lowered the rate of proliferation only when a high RII level was observed. An antibody against the anchoring region of RIIalpha showed cell cycle-dependent binding in contrast to antibodies against other regions, possibly related to altered binding to A-kinase anchoring protein. Following activation of PKA, relocalization of RIIalpha was confirmed by immunocytochemistry. In conclusion, it appears that activation of PKA II is important in regulation of alveolar type 2 cell proliferation.     

cAMP-dependent protein kinase is essential for hypoxia-mediated epithelial–mesenchymal transition,migration, and invasion in lung cancer cells

Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial–mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor β1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.     

Control of PKA stability and signalling by the RING ligase praja2

Activation of G-protein-coupled receptors (GPCRs) mobilizes compartmentalized pulses of cyclic AMP. The main cellular effector of cAMP is protein kinase A (PKA), which is assembled as an inactive holoenzyme consisting of two regulatory (R) and two catalytic (PKAc) subunits. cAMP binding to R subunits dissociates the holoenzyme and releases the catalytic moiety, which phosphorylates a wide array of cellular proteins. Reassociation of PKAc and R components terminates the signal. Here we report that the RING ligase praja2 controls the stability of mammalian R subunits. Praja2 forms a stable complex with, and is phosphorylated by, PKA. Rising cAMP levels promote praja2-mediated ubiquitylation and subsequent proteolysis of compartmentalized R subunits, leading to sustained substrate phosphorylation by the activated kinase. Praja2 is required for efficient nuclear cAMP signalling and for PKA-mediated long-term memory. Thus, praja2 regulates the total concentration of R subunits, tuning the strength and duration of PKA signal output in response to cAMP.     

Anti-proliferative effects of 8-chloro-cAMP and other cAMP analogs are unrelated to their effects on protein kinase A regulatory subunit expression

Conflicting reports have attributed 8-chloro-cAMP (Cl-cAMP)-mediated inhibition of tumor cell growth to either a toxic 8-chloro-adenosine (Cl-AdR) breakdown product or a Cl-cAMP-mediated decrease in ratio of Type I to Type II regulatory (R) subunits of protein kinase A (PKA). Using the MCF-7 human breast cancer and S49 mouse lymphoma cell lines as models, we show that the effects of Cl-cAMP and other cAMP analogs on growth and R subunit expression are unrelated. MCF-7 cell growth was insensitive to most analogs and inducers of cAMP, but was potently inhibited by Cl-cAMP acting through uptake and phosphorylation of its Cl-AdR breakdown product. Possible roles of adenosine receptors or P(2) purinoceptors in these Cl-cAMP-mediated growth effects were ruled out by studies with agonists and antagonists. Cholera toxin markedly decreased the ratio of Type I to Type II R subunits in MCF-7 cells without affecting growth, while growth inhibitory concentrations of Cl-cAMP or Cl-AdR had insignificant effects on this ratio. In S49 cells, where PKA activation is known to inhibit cell growth, PKA-deficient mutants retained sensitivity to both Cl-cAMP and the related 8-bromo-cAMP. Adenosine kinase (AK)-deficient S49 cells were inhibited only by higher concentrations of these 8-halogenated cAMP analogs. Of the commonly used cAMP analogs, only 8-(4-chlorophenylthio)-cAMP acted purely as a cyclic nucleotide-having no effect on PKA-deficient cells, but strongly inhibiting both wild-type and AK-deficient cells. Where growth inhibitory concentrations of most cAMP analogs reduced RI expression in the AK-deficient mutant, a functionally equivalent concentration of (N(6), O(2'))dibutyryl-cAMP maintained or increased this expression.     

Protein kinase A subunit expression is altered in Bloom syndrome fibroblasts and the BLM protein is increased in adrenocortical hyperplasias: inverse findings for BLM and PRKAR1A.

Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.     

Norepinephrine specifically stimulates ribonucleotide reductase subunit R2 gene expression in proliferating brown adipocytes: mediation via a cAMP/PKA pathway involving Src and Erk1/2 kinases

We have examined whether a qualitative switch occurs in the response of the ribonucleotide reductase (RNR) genes to the effect of the physiological cAMP-elevating agent norepinephrine (NE) during the development of brown adipocytes. Basal expression of the genes for both RNR subunits, R1 and R2, was high in proliferating cells, but was markedly down-regulated in parallel with adipocyte differentiation. NE stimulation, which promotes DNA synthesis and proliferation of brown preadipocytes, resulted in an increased expression of the R2 gene in proliferating cells (1.6-fold), but was without effect on R1 expression. In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression. The NE stimulation of R2 expression in preadipocytes was mimicked by forskolin and abolished by H89, demonstrating mediation via cAMP and protein kinase A (PKA). Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression. We conclude that adrenergic stimulation of brown adipocytes by NE specifically elevates expression of the RNR subunit R2 gene in the proliferative stage of brown adipocyte development, the mediating pathway being a cAMP/PKA cascade further involving Src and the MAP kinase Erk1/2. These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status.     

Molecular basis for isoform-specific autoregulation of protein kinase A

Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P(0)-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P(0)-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P(0), whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P(0) from serine to alanine. In contrast, PKA-Calpha forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P(0): Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells.     

Desensitization of the Y1 cell adrenocorticotropin receptor: evidence for a restricted heterologous mechanism implying a role for receptor-effector complexes

Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness. Using the mouse adrenocortical Y1 cell line we demonstrate that ACTH effectively desensitizes the cAMP response of its own receptor, the melanocortin 2 receptor (MC2R), in these cells with a maximal effect between 30 and 60 min. Neither forskolin nor isoproterenol (in Y1 cells stably transfected with the beta(2)-adrenergic receptor) desensitize this ACTH response. ACTH desensitizes its receptor at concentrations at which only a fraction of receptors are occupied, implying that this mechanism acts on agonist-unoccupied receptors. Y1 cells express G protein-coupled receptor kinase (GRK) 2 and 5, but stable expression of a dominant negative GRK2 (K220W) only marginally reduces the desensitization by ACTH. The protein kinase A (PKA) inhibitor, H89, extinguishes almost the entire desensitization response over the initial 30-min period at all concentrations of ACTH. A mutant MC2R in which the single consensus PKA phosphorylation site has been mutated (S208A) when expressed in MC2R-negative Y6 cells is also unable to desensitize. These data imply a heterologous, PKA-dependent, mode of desensitization, which is restricted to agonist-occupied and -unoccupied MC2R, possibly as a consequence of receptor/effector complexes that functionally compartmentalize this receptor.     

Crystal structures of RIalpha subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3',5'-cyclic monophosphothioate and (Sp)-adenosine 3',5'-cyclic monophosphothioate, the phosphothioate analogues of cAMP

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced. This strand forms an intermolecular antiparallel beta-sheet with the same strand in an adjacent molecule and implies that the RIalpha subunit can form a weak homodimer even in the absence of its dimerization domain.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.     

Identification and characterization of novel PKA holoenzymes in human T lymphocytes

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

PKA compartmentalization links cAMP signaling and autophagy

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.Subject terms: Kinases, Autophagy     

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.     

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.     

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.