Altered cAMP-dependent protein kinase subunit immunolabeling in post-mortem brain from patients with bipolar affective disorder

Previous findings of reduced [3H]cAMP binding and increased activities of cAMP-dependent protein kinase (PKA) in discrete post-mortem brain regions from patients with bipolar affective disorder (BD) suggest that PKA, the major downstream target of cAMP, is also affected in this illness. As prolonged elevation of intracellular cAMP levels can modify PKA regulatory (R) and catalytic (C) subunit levels, we sought to determine whether these PKA abnormalities are related to changes in the abundance of PKA subunits in BD brain. Using immunoblotting techniques along with PKA subunit isoform-specific polyclonal antisera, levels of PKA RIalpha, RIbeta, RIIalpha, RIIbeta and Calpha subunits were measured in cytosolic and particulate fractions of temporal, frontal and parietal cortices of post-mortem brain from BD patients and matched, non-neurological, non-psychiatric controls. Immunoreactive levels of cytosolic Calpha in temporal and frontal cortices, as well as that of cytosolic RIIbeta in temporal cortex, were significantly higher in the BD compared with the matched control brains. These changes were independent of age, post-mortem interval or pH and unrelated to ante-mortem lithium treatment or suicide. These findings strengthen further the notion that the cAMP/PKA signaling system is up-regulated in discrete cerebral cortical regions in BD.     

Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.     

Isoform specific differences in binding of a dual-specificity A-kinase anchoring protein to type I and type II regulatory subunits of PKA

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMP-dependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIalpha than to RIalpha. The higher affinity for RIIalpha was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIalpha and RIIalpha was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIalpha was independent of solution ionic strength, whereas RIIalpha had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIalpha but not to RIIalpha. These isoform-specific differences provide a foundation for developing isoform-specific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring.     

Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.     

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.     

Differential localization of protein kinase A type II isozymes in the Golgi-centrosomal area.

Selectivity in the action of cAMP may be mediated by compartmentalized pools of cyclic AMP-dependent protein kinase (PKA). PKA type II is directed to different subcellular loci by interaction of the type II regulatory subunits (RIIalpha, RIIbeta) with A-kinase anchoring proteins. In order to separately investigate the subcellular localization of PKA type II isozymes, monospecific antibodies to human RIIalpha and RIIbeta subunits of PKA were developed. We demonstrate that centrosomes bind both RIIalpha and RIIbeta. Centrosomes were the preferred intracellular anchoring site for RIIbeta. However, centrosomal localization of RIIbeta was observed only in some highly differentiated cells such as keratinocytes, granulosa cells, and macrophages and in all neoplastic cell lines examined. Centrosomal localization of RIIbeta was not observed in normal undifferentiated cells such as fibroblasts, myoblasts, and T and B cells. In contrast, RIIalpha was abundant in the Golgi area and in the trans-Golgi network (TGN). Furthermore, although RIIalpha appeared to colocalize with microtubules in the Golgi/TGN, extractions with nonionic detergent demonstrated that RIIalpha was mainly membrane-associated. In addition, alterations of microtubule dynamics with Nocodazole or Taxol affected the distribution of the detergent-extractable pool of RIIalpha, indicating that RIIalpha may localize with microtubule-associated vesicles. Thus, RIIalpha and RIIbeta clearly localize differently in the Golgi-centrosomal region. This indicates specific roles for PKA isozymes containing either RIIalpha or RIIbeta.     

Related protein-protein interaction modules present drastically different surface topographies despite a conserved helical platform

The subcellular localization of cAMP-dependent protein kinase (PKA) occurs through interaction with A-Kinase Anchoring Proteins (AKAPs). AKAPs bind to the PKA regulatory subunit dimer of both type Ialpha and type IIalpha (RIalpha and RIIalpha). RIalpha and RIIalpha display characteristic localization within different cell types, which is maintained by interaction of AKAPs with the N-terminal dimerization and docking domain (D/D) of the respective regulatory subunit. Previously, we reported the solution structure of RIIa D/D module, both free and bound to AKAPs. We have now solved the solution structure of the dimerization and docking domain of the type Ialpha regulatory dimer subunit (RIalpha D/D). RIalpha D/D is a compact docking module, with unusual interchain disulfide bonds that help maintain the AKAP interaction surface. In contrast to the shallow hydrophobic groove for AKAP binding across the surface of the RIIalpha D/D dimeric interface, the RIalpha D/D module presents a deep cleft for proposed AKAP binding. RIalpha and RIIalpha D/D interaction modules present drastically differing dimeric topographies, despite a conserved X-type four-helix bundle structure.     

Substrate enhances the sensitivity of type I protein kinase a to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.     

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.     

Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions

The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.     

Involvement of type I protein kinase A in the differentiation of L6 myoblast in conjunction with phosphatidylinositol 3-kinase

To investigate the role of protein kinase A (PKA) (EC 2.7.1.37) in myogenesis, PKA activity was closely monitored during the differentiation of L6 rat skeletal myoblasts. As the differentiation proceeded, total PKA activity increased about 2-3 fold, and the protein levels of PKA RIalpha and Calpha subunits increased about 3-4 fold. We then looked at the effect of the specific inhibitor for PKA, N-[2-(p-bromocinnamy-lamino)-ethyl]-5-isoquinoline-sulfonamide (H89), on the differentiation of L6 myoblasts. H89 completely blocked the myotube formation and abolished the up-regulation of RIalpha and Ca. This inhibitory effect of H89 was dose-dependent and could be reversed upon removal of H89 from the culture medium. Furthermore, we demonstrated that specific inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin, and LY294002 blocked the myotube formation and abolished the increase of PKA activity, which normally accompanied the differentiation of myoblasts. These results suggest that type I PKA may play a functional role(s) in the differentiation of myoblast as a putative downstream effector of the PI3K signaling pathway.     

A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dependent protein kinase

A kinase-anchoring proteins (AKAPs) target PKA to specific microdomains by using an amphipathic helix that docks to N-terminal dimerization and docking (D/D) domains of PKA regulatory (R) subunits. To understand specificity, we solved the crystal structure of the helical motif from D-AKAP2, a dual-specific AKAP, bound to the RIIalpha D/D domain. The 1.6 Angstrom structure reveals how this dynamic, hydrophobic docking site is assembled. A stable, hydrophobic docking groove is formed by the helical interface of two RIIalpha protomers. The flexible N terminus of one protomer is then recruited to the site, anchored to the peptide through two essential isoleucines. The other N terminus is disordered. This asymmetry provides greater possibilities for AKAP docking. Although there is strong discrimination against RIalpha in the N terminus of the AKAP helix, the hydrophobic groove discriminates against RIIalpha. RIalpha, with a cavity in the groove, can accept a bulky tryptophan, whereas RIIalpha requires valine.     

Deletion of type IIalpha regulatory subunit delocalizes protein kinase A in mouse sperm without affecting motility or fertilization.

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.     

cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIbeta and two mutant RIIbeta subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a Ka identical to the Kd(cAMP) of full-length RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.     

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.     

Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange

The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIalpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIalpha compared to RIIalpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.     

Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo

A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.