Ghrelin is related to lower brain reward activation during touch.

The gut hormone ghrelin drives food motivation and increases food intake, but it is also involved in the anticipation of and response to rewards other than food. This pre-registered study investigated how naturally varying ghrelin concentrations affect the processing of touch as a social reward in humans. Sixty-seven volunteers received slow caressing touch (so-called CT-targeted touch) as a social reward and control touch on their shins during 3T functional imaging on two test days. On one occasion, participants were fasted, and on another, they received a meal. On each occasion, plasma ghrelin was measured at three time points. All touch was rated as more pleasant after the meal, but there was no association between ghrelin concentrations and pleasantness. CT-targeted touch was rated as the most pleasant and activated somatosensory and reward networks (whole brain). A region-of-interest in the right medial orbitofrontal cortex (mOFC) showed lower activation during all touches, the higher the ghrelin concentrations were. During CT-targeted touch, a larger satiety response (ghrelin decrease after the meal) was associated with higher mOFC activation, and this mOFC activation was associated with higher experienced pleasantness. Overall, higher ghrelin concentrations appear to be related to a lower reward value for touch. Ghrelin may reduce the value of social stimuli, such as touch, to promote food search and intake in a state of low energy. This suggests that the role of ghrelin goes beyond assigning value to food reward.

Serum albumin and HCO3- regulate separate pools of ATP in human spermatozoa.

STUDY QUESTION: Do the known capacitating agents HCO(3)(-) and serum albumin regulate the generation of ATP required for sperm motility and capacitation? SUMMARY ANSWER: Serum albumin and HCO(3)(-) seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa. WHAT IS KNOWN ALREADY: Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation. STUDY DESIGN, SIZE, DURATION: Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO(3)(-) and serum albumin for up to 4 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO(3)(-) were analysed by metabolic tracing of (13)C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions. MAIN RESULTS AND THE ROLE OF CHANCE: We found that HCO(3)(-) significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO(3)(-)-induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl- and methyl-ß-cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca(2+) channel CatSper. Finally, we demonstrated that neither HCO(3)(-) nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO(3)(-). LIMITATIONS, REASONS FOR CAUTION: Great variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species. WIDER IMPLICATIONS OF THE FINDINGS: It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO(3)(-) stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques. STUDY FUNDING: The work was funded by Spermatech AS, The University of Oslo and the Research Council of Norway. COMPETING INTEREST(S): T.H.H. and K.R.R. are employees at Spermatech. B.S.S is a shareholder in Spermatech.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

BACKGROUND: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Lipid-lowering and anti-inflammatory effects of tetradecylthioacetic acid in HIV-infected patients on highly active antiretroviral therapy.

BACKGROUND: Highly active antiretroviral therapy (HAART) often leads to a dramatic improvement in clinical, viral and immunologic parameters in HIV-infected individuals. However, the emergence of long-term side-effects of HAART and in particular dylipidaemia is increasingly reported. Based on the potential lipid-lowering and immunomodulatory properties of tetradecylthioacetic acid (TTA) we examined whether TTA in combination with dietary intervention could modify lipid levels in peripheral blood in HIV-infected patients on HAART. MATERIALS AND METHODS: Ten HIV-infected patients on protease inhibitor-based HAART with hyperlipidaemia followed a cholesterol-lowering diet throughout the study period (8 weeks). During the last 4 weeks of the study all patients received TTA (1 g qd) in addition to the cholesterol-lowering diet. RESULTS: Our main and novel findings were: (i) TTA in combination with dietary intervention reduces total cholesterol, LDL cholesterol, triglycerides and LDL/HDL cholesterol in these patients, and a particularly suppressing effect was observed during the TTA phase regarding total cholesterol. (ii) During the TTA phase, the cholesterol-lowering effect was accompanied by a significant reduction in plasma levels of tumour necrosis factor alpha. (iii) Our studies in peripheral blood mononuclear cells from these patients and in the liver from wild-type mice receiving TTA suggest that the hypolipidaemic effects of TTA may involve up-regulation of scavenger and LDL-receptor expression. CONCLUSIONS: Although few patients were studied, the present pilot study suggests that TTA combined with dietary intervention could be an interesting therapeutic approach in HIV-infected patients on HAART, potentially resulting in both hypolipidaemic and anti-inflammatory effects.

CDK1-mediated phosphorylation of the RIIalpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIalpha from centrosomes at mitosis.

Protein kinase A regulatory subunit RIIalpha is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34(cdc2) kinase (CDK1) has been shown to phosphorylate RIIalpha on T54 and this has been proposed to alter the subcellular localization of RIIalpha. We have made stable transfectants from an RIIalpha-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIalpha (RIIalpha(T54E)). When expressed, RIIalpha detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIalpha(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIalpha is solubilized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N-terminal RII-binding domain of AKAP450, whereas small neutral residues do not change affinity detected by surface plasmon resonance. In addition, only RIIalpha(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIalpha(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIalpha transfectants. Our results suggest that T54 phosphorylation of RIIalpha by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.

Differential expression and regulation of the PKA signalling pathway in fast and slow skeletal muscle.

To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.

Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

HA95 is a protein of the chromatin and nuclear matrix regulating nuclear envelope dynamics.

We report a role for HA95, a nuclear protein with high homology to the nuclear A-kinase anchoring protein AKAP95, in the regulation of nuclear envelope-chromatin interactions. Biochemical and photobleaching data indicate that HA95 is tightly associated with chromatin and the nuclear matrix/lamina network in interphase, and bound to chromatin at mitosis. HA95 resides in a complex together with lamin B receptor (LBR), lamina-associated polypeptide (LAP)2 and emerin, integral proteins of the inner nuclear membrane. Cross-linking experiments, however, illustrate a tight association of HA95 with LBR and LAP2 only. Intra-nuclear blocking of HA95 with anti-HA95 antibodies abolishes nuclear breakdown in a mitotic HeLa cell extract. The antibodies inhibit nuclear membrane breakdown and chromatin condensation - the latter independently of nuclear membranes. However, lamina disassembly is not affected, as judged by immunological analyses of A/C- and B-type lamins. In contrast, immunoblocking of HA95 bound to condensed chromosomes does not impair chromatin decondensation, nuclear membrane reassembly or lamina reformation. Our results argue for a role for HA95 in anchoring nuclear membranes and lamins to chromatin in interphase, and in releasing membranes from chromatin at mitosis. The data also suggest that HA95 is not involved in initial binding of membranes to chromatin upon nuclear reassembly. We propose that HA95 is a central platform at the chromatin/nuclear matrix interface implicated in regulating nuclear envelope-chromatin interactions during the cell cycle.

Specificity in the cAMP/PKA signaling pathway. Differential expression,regulation, and subcellular localization of subunits of PKA.

A large number of hormones, neurotransmitters and other signal substances utilize adenosine 3',5' cyclic monophosphate (cAMP) as an intracellular second messenger. Cyclic AMP regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the cAMP-dependent protein kinase (PKA). The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP-engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we will describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece.

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s).

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may occur.

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95.

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.

PKAI as a potential target for therapeutic intervention.

We have mapped a molecular mechanism for the impaired T-cell function in HIV infection and common variable immunodeficiency (CVI). Protein kinase A type I (PKAI) has a key role as an inhibitor of immune function in T lymphocytes and is activated following antigen receptor triggering. T cells from patients with HIV infection and CVI have increased activation of PKAI. This inhibits immune function and proliferation of T cells. Selective antagonists that block cAMP action through PKAI improve the immune function of T cells from HIV-infected patients up to 300%. Furthermore, combination of cAMP antagonists with interleukin-2 normalized immune responses of T cells from all patients examined and stimulated immune function of T cells from HIV-infected patients up to 600%. In addition, in vitro experiments indicate that approximately 50% of patients with CVI have a T-cell dysfunction that might benefit from a treatment reversing PKAI hyperactivation. This outlines PKAI as a potentially attractive drug target for immunomodulating therapy in HIV infection, as well as for the treatment of other immunodeficiency disorders such as CVI.

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

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.

Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450.

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Increased activation of protein kinase A type I contributes to the T cell deficiency in common variable immunodeficiency.

The molecular mechanisms underlying the T cell dysfunction often present in common variable immunodeficiency (CVI) are not established. cAMP-dependent protein kinase A type I (PKAI) is an important inhibitor of T cell proliferation after Ag stimulation. We therefore investigated the possibility that activation of PKAI may be involved in the development of T cell dysfunction in CVI. An exogenously added PKAI-selective antagonist (Rp-8-Br-cAMPS) induced a significant increase in anti-CD3-stimulated PBMC proliferation in 20 CVI patients compared with no effect in 15 controls. Purified T cells from 7 CVI patients with strictly defined T cell deficiency had elevated endogenous cAMP levels compared with controls. Treatment of T cells from these CVI patients with Rp-8-bromo-cAMP-phosphorothioate markedly improved anti-CD3-stimulated proliferation (up to 3.7-fold), particularly in CD4+ lymphocytes, reaching proliferation levels comparable to control values. No effect of cAMP antagonist on T cell proliferation was seen in controls. In these CVI patients, cAMP antagonist also increased IL-2 production in anti-CD3-stimulated T cells. However, exogenously added IL-2 at concentrations comparable to the achieved increase in IL-2 levels after addition of cAMP antagonist had no effect on T cell proliferation. Furthermore, the stimulatory effects of exogenously added IL-2 at higher concentrations and cAMP antagonist on T cell proliferation were additive. Our findings indicate that increased PKAI activation may be an important molecular basis for the T cell defect in CVI and suggest that the cAMP/PKAI system may be a potential molecular target for immunomodulating therapy in these patients.