Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells.

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.

Extracellular cyclic adenosine monophosphate-dependent protein kinase A autoantibody and C-reactive protein as serum biomarkers for diagnosis of cancer in dogs.

Protein kinase A, a cyclic adenosine monophosphate (AMP)-dependent enzyme, normally exists within mammalian cells; however, in cancer cells, it can leak out and be found in the serum. Extracellular cyclic AMP-dependent protein kinase A (ECPKA) has been determined to increase in the serum of cancer-bearing dogs. However, there have been no reports in the veterinary literature on serum ECPKA autoantibody (ECPKA-Ab) expression in dogs with cancer. The aim of this study was to evaluate ECPKA-Ab and C-reactive protein (CRP) as serum biomarkers for cancer in dogs. ECPKA-Ab and CRP levels were detected by an enzyme-linked immunosorbent assay in serum samples from dogs with malignant tumours (n = 167), benign tumours (n = 42), or non-tumour disease (n = 155) and from healthy control dogs (n = 123). ECPKA-Ab and CRP levels were significantly higher in the dogs with malignant tumours than in those with benign tumours or non-tumour diseases, as well as in the healthy controls (P < 0.001, Kruskal-Wallis test). There was a significant positive correlation between the neoplastic index, which was developed using ECPKA-Ab and CRP levels, and the presence of cancer in dogs (P < 0.001); the area under the receiver-operating characteristic curve was estimated to be >0.85 (P < 0.001). In conclusion, ECPKA-Ab is a potential serum biomarker for a broad spectrum of cancers. Combined measurement of CRP and ECPKA-Ab levels in serum improves the sensitivity and accuracy of a diagnosis of cancer in dogs.

MyRIP anchors protein kinase A to the exocyst complex.

The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery.

Distribution of insoluble cAMP-dependent kinase type RI and RII in the lizard and turtle central nervous system.

cAMP is a universal second messenger. In eucaryotes it acts mainly via protein kinases composed of regulatory (R) and catalytic subunits; their subcellular distribution may differ according to the cell type. In rodent brain, peculiar detergent-insoluble RIalpha aggregates were previously described in neurons of areas related to the limbic system, while RIIbeta is more evenly distributed also in non-nervous cells. It is unclear whether the regional distribution of regulatory subunits is typical of mammalian brain. Western blots and immunohistochemistry showed that in lizard brains a large fraction of the cAMP-dependent protein kinase regulatory isoforms is insoluble, as in mammals. Insoluble RIalpha and RII regulatory isoforms were not evenly distributed but organized in clearly separated aggregates. Numerous RII aggregates were present in almost all brain regions and were found also in non-nervous cells. As shown by immunohistochemistry and equilibrium binding of fluorescently tagged cAMP, RIalpha aggregates were restricted to neurons of some brain regions: telencephalon, particularly medial cortical areas, dorsal ventricular ridge, olfactory pathways, medial hypothalamus and cerebellar granular layer were intensely labelled. A very weak RIalpha labelling was detected in the brainstem reticular formation, in the periaqueductal gray and in the spinal cord dorsal horn. A similar distribution of RIalpha aggregates was also found in turtle brains. Their distribution is reminiscent of that observed in mammals, although with some differences in relative intensity and persistence. The supramolecular organization of the RIalpha isoform may help in establishing homologies and differences between brain areas involved in visceroemotional control.

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.

Differential activity profile of cAMP-dependent protein kinase isoforms during long-term memory consolidation in the crab Chasmagnathus.

The isoforms of cAMP-dependent protein kinase (PKA) show distinct biochemical properties and subcellular localization, suggesting different physiological functions, and conferring the fine-tuning between the activation of cAMP-PKA cascade and the cellular response. The critical role of PKA in memory and synaptic plasticity has been extensively demonstrated both in vertebrates and invertebrates, but the role of PKA isoforms is a matter of debate. Here we present experimental data showing differential PKA activation profiles after two different experiences: an instance of associative contextual learning (context-signal learning) and a single exposure to a novel context, both in the learning and memory model of the crab Chasmagnathus. Differences were found in the temporal course of activation and in the involvement of PKA isoforms. We found increased PKA activity immediately and 6 h after context-signal training correlating with the critical periods during which pharmacological inhibition of PKA disrupts memory formation. In contrast, PKA activity increased immediately but not 6 h after single exposure to a novel context. The amounts of PKA I and PKA II holoenzymes were analyzed to determine changes in holoenzyme levels and/or differential activation induced by both experiences. Results indicate that context-induced PKA activation is at least in part due to PKA II, and that PKA activation 6 h after context-signal learning coincides with an increase in the total level of PKA I. Considering the higher sensitivity of PKA I to cAMP, its increment can account for the PKA activation found 6 h after training and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.

Chronic antidepressant treatment prevents accumulation of gsalpha in cholesterol-rich, cytoskeletal-associated, plasma membrane domains (lipid rafts).

Previous studies demonstrated that Gsalpha migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with the antidepressants desipramine and fluoxetine. Antidepressant treatment also causes a Gsalpha redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsalpha and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsalpha redistribution and the increased coupling between Gsalpha and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two fuctionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsalpha content of TTX-100 insoluble membrane domains by as much as 60%, while the inactive fluoxetine analog LY368514 had no effect. Disruption of these membrane domains with the cholesterol chelator methyl-beta-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsalpha localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsalpha out of detergent-resistant membrane domains in a manner identical to that seen with antidepressant treatment. The data presented here further substantiate the role of Gsalpha as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsalpha and the cytoskeleton is involved in this process.

Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway.

The gastrointestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is one of the most important regulators of insulin secretion following ingestion of a meal. GIP stimulates insulin secretion from the pancreatic beta-cell via its G protein-coupled receptor activation of adenylyl cyclase and other signal transduction pathways, but there is little known regarding subsequent protein kinase pathways that are activated. A screening technique was used to determine the relative abundance of 75 protein kinases in CHO-K1 cells expressing the GIP receptor and in two pancreatic beta-cell lines (betaTC-3 and INS-1 (832/13) cells). This information was used to identify kinases that are potentially regulated following GIP stimulation, with a focus on GIP regulation of the ERK1/2 MAPK pathway. In CHO-K1 cells, GIP induced phosphorylation of Raf-1 (Ser-259), Mek1/2 (Ser-217/Ser-221), ERK1/2 (Thr-202 and Tyr-204), and p90 RSK (Ser-380) in a concentration-dependent manner. Activation of ERK1/2 was maximal at 4 min and was cAMP-dependent protein kinase-dependent and protein kinase C-independent. Studies using a beta-cell line (INS-1 clone 832/13) corroborated these findings, and it was also demonstrated that the ERK1/2 module could be activated by GIP in the absence of glucose. Finally, we have shown that GIP regulation of the ERK1/2 module is via Rap1 but does not involve Gbetagamma subunits nor Src tyrosine kinase, and we propose that cAMP-based regulation occurs via B-Raf in both CHO-K1 and beta-cells. These results establish the importance of GIP in the cellular regulation of the ERK1/2 module and identify a role for cAMP in coupling its G protein-coupled receptors to ERK1/2 activity in pancreatic beta-cells.

Classification and phylogenetic analysis of the cAMP-dependent protein kinase regulatory subunit family.

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by beta-strands 6 and 7 of the crystal structures of bovine RIalpha and rat RIIbeta (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F-G-E-[LIV]-A-L-[LIMV]-x(3)-[PV]-R-[ANQV]-A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects alpha-helix B' and beta strand 7.

Selective regulation of G protein-coupled receptor-mediated signaling by G protein-coupled receptor kinase 2 in FRTL-5 cells: analysis of thyrotropin, alpha(1B)-adrenergic, and A(1) adenosine receptor-mediated responses.

G protein-coupled receptor kinases (GRKs) play a key role in the process of receptor homologous desensitization. In the present study, we address the question of whether a variety of receptors coupled to different G protein subtypes and naturally expressed on the same cell are selectively regulated by GRK2. The signaling stimulated by thyrotropin (TSH), alpha(1B)-adrenergic, and A(1) adenosine receptors was studied in FRTL-5 cells permanently transfected to overexpress GRK2 and GRK2-K220R, a kinase dead GRK dominant negative mutant. In FRTL-5 overexpressing GRK2, TSH-induced cyclic AMP response was attenuated, indicating that TSH receptor is desensitized by this kinase. Consistently, FRTL-5 cells overexpressing GRK2-K220R show increased TSH-induced cyclic AMP response, demonstrating that this receptor is under tonic control by GRK. Unlike TSH receptor, alpha(1B)-adrenergic receptor response was unaffected in FRTL-5 overexpressing GRK2 and GRK2-K220R. When A(1) adenosine receptors were stimulated, G(ialpha)-mediated cyclic AMP inhibition was totally unaffected by overexpression of either GRK2 or GRK2-K220R. By contrast, G(betagamma)-mediated response (activation of mitogen-activated protein kinases) was efficiently desensitized by GRK2 but was unaffected by GRK2-K220R overexpression. The present study documents that overexpression of GRK2 results in a selective regulation of different G protein-coupled receptors expressed on the same cell and that this kinase can regulate preferentially only one of the different pathways activated by the same receptor. The preferential regulation of the A(1) adenosine receptor-stimulated mitogen-activated protein kinases by GRK2 indicates that this kinase can have additional regulatory effects on G(betagamma)-stimulated pathways, possibly through direct binding and regulation of the receptor-G(betagamma) complex.

Purification of a regulatory subunit of type II cAMP-dependent protein kinase from Drosophila heads.

The cytosolic extract from Drosophila heads was separated using anion-exchange column chromatography. Two types of cAMP-dependent protein kinase (PKA), type I and type II, were detected, and type II PKA was found to be a major isozyme. The regulatory subunit of type II PKA (RII) was purified, and only one isoform was observed. The purified protein had an apparent molecular mass of 51 kDa on SDS gel electrophoresis. Partial amino acid sequences of the protein were almost identical with the RII alpha subunit of human. Since PKA has been implicated to be especially important for learning and memory in Drosophila, the RII subunit may play an essential role in the regulation of neuronal activity in the brain of Drosophila, and possibly in human.

Regulation of G protein-coupled receptor kinase subtypes in activated T lymphocytes. Selective increase of beta-adrenergic receptor kinase 1 and 2.

Beta-adrenergic receptor kinase (beta ARK) is a serine-threonine kinase involved in the process of homologous desensitization of G-coupled receptors. beta ARK is a member of a multigene family, consisting of six known subtypes, also named G protein-coupled receptor kinases (GRK 1-6). In this study we investigated the expression of GRKs during the process of T cell activation, which is of fundamental importance in regulating immune responses. T cell activation was induced by exposing mononuclear leukocytes (MNL) to PHA and confirmed by tritiated thymidine incorporation measurement. A substantial increase of GRK activity (as measured by in vitro phosphorylation of rhodopsin) was found after 48 h (331 +/- 80% of controls) and 72 h (347 +/- 86% of controls) of exposure to PHA. A threefold increase of beta ARK1 immunoreactivity was found in MNL exposed to PHA for 72 h. Persistent activation of protein kinase C (PKC) by 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was able to increase beta ARK activity to the same extent as PHA, suggesting a PKC-mediated mechanism. The kinetic of beta-adrenergic-stimulated cAMP production was substantially modified in TPA and PHA-activated cells, indicating that the increased GRK activity resulted in an increased beta-adrenergic homologous desensitization. A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h. A significant increase in beta ARK1 and beta ARK2 mRNA expression was observed 48 h after mitogen stimulation, while mRNA expression of GRK5 and GRK6 was not changed. In conclusion our data show that the expression of GRK subtypes is actively and selectively modulated according to the functional state of T lymphocytes.