Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells.

Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and ß-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation.

Blau syndrome-associated Nod2 mutation alters expression of full-length NOD2 and limits responses to muramyl dipeptide in knock-in mice.

The biochemical mechanism by which mutations in nucleotide-binding oligomerization domain containing 2 (NOD2) cause Blau syndrome is unknown. Several studies have examined the effect of mutations associated with Blau syndrome in vitro, but none has looked at the implication of the mutations in vivo. To test the hypothesis that mutated NOD2 causes alterations in signaling pathways downstream of NOD2, we created a Nod2 knock-in mouse carrying the most common mutation seen in Blau syndrome, R314Q (corresponding to R334Q in humans). The endogenous regulatory elements of mouse Nod2 were unaltered. R314Q mice showed reduced cytokine production in response to i.p. and intravitreal muramyl dipeptide (MDP). Macrophages from R314Q mice showed reduced NF-κB and IL-6 responses, blunted phosphorylation of MAPKs, and deficient ubiquitination of receptor-interacting protein 2 in response to MDP. R314Q mice expressed a truncated 80-kDa form of NOD2 that was most likely generated by a posttranslational event because there was no evidence for a stop codon or alternative splicing event. Human macrophages from two patients with Blau syndrome also showed a reduction of both cytokine production and phosphorylation of p38 in response to MDP, indicating that both R314Q mice and cells from patients with Blau syndrome show reduced responses to MDP. These data indicate that the R314Q mutation when studied with the Nod2 endogenous regulatory elements left intact is associated with marked structural and biochemical changes that are significantly different from those observed from studies of the mutation using overexpression, transient transfection systems.

Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity.

The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme. Its primary components are A-kinase anchoring proteins (AKAPs) 3 and 4, which suggests that the FS affects flagellar beating via the scaffolding of signaling pathways necessary for motility. Sperm proteins ROPN1 and ROPN1L bind AKAP3. To determine the role of ROPN1 and ROPN1L in sperm function, we created mice deficient in ROPN1 (RKO), mice deficient in ROPN1L (RLKO), and double knockout mice (DKO). All three strains of mice had normal testicular morphology and spermatogenesis. Only the DKOs had obvious defects in sperm morphology (thinning and shredding of the principal piece), which was accompanied by a reduction in AKAP3 levels. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. RKO mice had moderately impaired motility and increased levels of ROPN1L. DKO sperm were immotile. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. Together these data indicate that ROPN1L and ROPN1 compensate for each other in the absence of the opposing protein, possibly to maintain AKAP3 incorporation in the FS. Sperm from mice lacking ROPN1L exhibited reductions in both cAMP-dependent protein kinase (PKA) phosphorylation of a 270-kDa protein (perhaps FSCB), and in capacitation-induced tyrosine phosphorylation. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. These data demonstrate that mutations in ROPN1 and ROPN1L can cause defects in FS integrity, sperm motility, and PKA-dependent signaling processes, leading to male infertility.

α-Tocopherol (vitamin E) stimulates cyclic AMP production in human peripheral mononuclear cells and alters immune function.

α-Tocopherol, the most biologically active member of the vitamin E family of fat soluble compounds, exhibits both antioxidant and anti-inflammatory properties. However, its mechanisms of action are not fully understood. Here, we show that, unlike other antioxidants, α-tocopherol stimulates the production of cyclic adenosine monophosphate (cAMP). Inhibitor studies demonstrate that the prostaglandin EP2 and EP4 receptors and adenylyl cyclases mediate the effects of α-tocopherol on cAMP production. Additionally, we show that α-tocopherol attenuates pro-inflammatory cytokine and chemokine production. This study provides novel evidence that α-tocopherol stimulates cAMP signaling, suggesting a mechanism of action for the immunomodulatory effects of vitamin E.

PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells.

Controlled maturation of ovarian follicles is necessary for fertility. Follicles are restrained at an immature stage until stimulated by FSH secreted by pituitary gonadotropes. FSH acts on granulosa cells within the immature follicle to inhibit apoptosis, promote proliferation, stimulate production of steroid and protein hormones, and induce ligand receptors and signaling intermediates. The phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) pathway is a pivotal signaling corridor necessary for transducing the FSH signal. We report that protein kinase A (PKA) mediates the actions of FSH by signaling through multiple targets to activate PI3K/AKT. PKA uses a route that promotes phosphorylation of insulin receptor substrate-1 (IRS-1) on Tyr(989), a canonical binding site for the 85-kDa regulatory subunit of PI3K that allosterically activates the catalytic subunit. PI3K activation leads to activation of AKT through phosphorylation of AKT on Thr(308) and Ser(473). The adaptor growth factor receptor bound protein 2-associated binding protein 2 (GAB2) is present in a preformed complex with PI3K heterodimer and IRS-1, it is an A-kinase anchoring protein that binds the type I regulatory subunit of PKA, and it is phosphorylated by PKA on Ser(159). Overexpression of GAB2 enhances FSH-stimulated AKT phosphorylation. GAB2, thus, seems to coordinate signals from the FSH-stimulated rise in cAMP that leads to activation of PI3K/AKT. The ability of PKA to commandeer IRS-1 and GAB2, adaptors that normally integrate receptor/nonreceptor tyrosine kinase signaling into PI3K/AKT, reveals a previously unrecognized route for PKA to activate a pathway that promotes proliferation, inhibits apoptosis, enhances translation, and initiates differentiation of granulosa cells.

Loss of ASP but not ROPN1 reduces mammalian ciliary motility.

Protein kinase A (PKA) signaling is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins [AKAP-associated sperm protein (ASP), ropporin (ROPN1), sperm protein 17 (SP17) and calcium binding tyrosine-(Y)-phosphorylation regulated protein (CABYR)] share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease.

Retinoid-induced expression and activity of an immediate early tumor suppressor gene in vascular smooth muscle cells.

Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE) located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12ß) in cultured smooth muscle cells (SMC) as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12ß is a retinoid-induced, immediate-early gene. Akap12ß promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA) regulatory subunit overlay assays in SMC suggest a physical association between AKAP12ß and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12ß attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.

AKAPs in lipid rafts are required for optimal antigen presentation by dendritic cells.

Dendritic cell (DC) maturation and antigen presentation are regulated by activation of protein kinase A (PKA) signaling pathways, through unknown mechanisms. We have recently shown that interfering with PKA signaling through the use of anchoring inhibitor peptides hinders antigen presentation and DC maturation. These experiments provide evidence that DC maturation and antigen presentation are regulated by A-kinase anchoring proteins (AKAPs). Herein, we determine that the presence of AKAPs and PKA in lipid rafts regulates antigen presentation. Using a combination of western blotting and immuno-cytochemistry, we illustrate the presence of AKAP149, AKAP79, Ezrin and the regulatory subunits of PKA in DC lipid rafts. Incubation of DCs with the type II anchoring inhibitor, AKAP-in silico (AKAP-IS), removes Ezrin and RII from the lipid raft without disrupting raft formation. Addition of a lipid raft disruptor, methyl-ß-cyclodextrin, blocks the efficacy of AKAP-IS, suggesting that the lipid raft must be intact for AKAP-IS to inhibit antigen presentation. Ezrin and AKAP79 are present in the lipid raft of stimulated KG1 cells, but Ezrin is not present in the lipid raft of unstimulated KG1 cells and AKAP79 levels are greatly diminished, suggesting that Ezrin and AKAP79 may be the key AKAPs responsible for regulating antigen presentation.

Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms.

Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the ß-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.

Lipoic acid attenuates inflammation via cAMP and protein kinase A signaling.

BACKGROUND: Abnormal regulation of the inflammatory response is an important component of diseases such as diabetes, Alzheimer's disease and multiple sclerosis (MS). Lipoic acid (LA) has been shown to have antioxidant and anti-inflammatory properties and is being pursued as a therapy for these diseases. We first reported that LA stimulates cAMP production via activation of G-protein coupled receptors and adenylyl cyclases. LA also suppressed NK cell activation and cytotoxicity. In this study we present evidence supporting the hypothesis that the anti-inflammatory properties of LA are mediated by the cAMP/PKA signaling cascade. Additionally, we show that LA oral administration elevates cAMP levels in MS subjects. METHODOLOGY/PRINCIPAL FINDINGS: We determined the effects of LA on IL-6, IL-17 and IL-10 secretion using ELISAs. Treatment with 50 µg/ml and 100 µg/ml LA significantly reduced IL-6 levels by 19 and 34%, respectively, in T cell enriched PBMCs. IL-17 levels were also reduced by 35 and 50%, respectively. Though not significant, LA appeared to have a biphasic effect on IL-10 production. Thymidine incorporation studies showed LA inhibited T cell proliferation by 90%. T-cell activation was reduced by 50% as measured by IL-2 secretion. Western blot analysis showed that LA treatment increased phosphorylation of Lck, a downstream effector of protein kinase A. Pretreatment with a peptide inhibitor of PKA, PKI, blocked LA inhibition of IL-2 and IFN gamma production, indicating that PKA mediates these responses. Oral administration of 1200 mg LA to MS subjects resulted in increased cAMP levels in PBMCs four hours after ingestion. Average cAMP levels in 20 subjects were 43% higher than baseline. CONCLUSIONS/SIGNIFICANCE: Oral administration of LA in vivo resulted in significant increases in cAMP concentration. The anti-inflammatory effects of LA are mediated in part by the cAMP/PKA signaling cascade. These novel findings enhance our understanding of the mechanisms of action of LA.

Myeloid translocation gene 16b is a dual A-kinase anchoring protein that interacts selectively with plexins in a phospho-regulated manner.

The myeloid translocation gene (MTG) homologue Nervy associates with PlexinA on the plasma membrane, where it functions as an A-kinase anchoring protein (AKAP) to modulate plexin-mediated semaphorin signaling in Drosophila. Mammalian MTG16b is an AKAP found in immune cells where plexin-mediated semaphorin signaling regulates immune responses. This study provides the first evidence that MTG16b is a dual AKAP capable of binding plexins. These interactions are selective (PlexinA1 and A3 bind MTG, while PlexinB1 does not) and can be regulated by PKA-phosphorylation. Collectively, these data suggest a possible mechanism for the targeting and integration of adenosine 3',5'-cyclic monophosphate (cAMP) and semaphorin signaling in immune cells.

A-kinase anchoring in dendritic cells is required for antigen presentation.

BACKGROUND: Dendritic cells (DC) are the most potent antigen presenting cells (APC) of the immune system. Prostaglandin E(2), cyclic AMP, and protein kinase A (PKA) have all been shown to regulate DC maturation and activity. In other cells, the ability of these molecules to convey their signals has been shown to be dependent on A-kinase anchoring proteins (AKAPs). Here we present evidence for the existence and functional importance of AKAPs in human DC. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence and/or western analyses we identify AKAP79, AKAP149, AKAP95, AKAP LBC and Ezrin. We also demonstrate by western analysis that expression of AKAP79, AKAP149 and RII are upregulated with DC differentiation and maturation. We establish the functional importance of PKA anchoring in multiple aspects of DC biology using the anchoring inhibitor peptides Ht31 and AKAP-IS. Incubation of protein or peptide antigen loaded DC with Ht31 or AKAP-IS results in a 30-50% decrease in antigen presentation as measured by IFN-gamma production from antigen specific CD4(+) T cells. Incubation of LPS treated DC with Ht31 results in 80% inhibition of TNF-alpha and IL-10 production. Ht31 slightly decreases the expression of CD18 and CD11a and CD11b, slightly increases the basal expression of CD83, dramatically decreases the LPS stimulated expression of CD40, CD80 and CD83, and significantly increases the expression of the chemokine receptor CCR7. CONCLUSIONS: These experiments represent the first evidence for the functional importance of PKA anchoring in multiple aspects of DC biology.

Lipoic acid: a novel therapeutic approach for multiple sclerosis and other chronic inflammatory diseases of the CNS.

The naturally occurring antioxidant lipoic acid (LA) was first described as an essential cofactor for the conversion of pyruvate to Acetyl-CoA, a critical step in respiration. LA is now recognized as a compound that has many biological functions. Along with its reduced form dihydrolipoic acid (DHLA), LA reduces and recycles cellular antioxidants such as glutathione, and chelates zinc, copper and other transition metal ions in addition to heavy metals. LA can also act as a scavenger of reactive oxygen and nitrogen species. By acting as an insulin mimetic agent, LA stimulates glucose uptake in many different cell types and can also modulate insulin signaling. The p38 and ERK MAP kinase pathways, AKT and NFkappaB are all regulated by LA. In addition, LA activates the prostaglandin EP2 and EP4 receptors to stimulate the production of the small molecule cyclic adenosine 5' monophosphate (cAMP). These diverse actions suggest that LA may be therapeutically effective in treating oxidative stress associated diseases. This review discusses the known biochemical properties of LA, its antioxidant properties, its ability to modulate signal transduction pathways, and the recent progress made in the utilization of LA as a therapeutic alternative for multiple sclerosis, Alzheimer's disease and diabetic neuropathy.

Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells.

The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells in a dose-dependent manner. Studies using pharmacological inhibitors and receptor transfection experiments indicate that LA stimulates cAMP production via activation of the EP2 and EP4 prostanoid receptors and adenylyl cyclase. In addition, LA suppressed interleukin (IL)-12/IL-18 induced IFNgamma secretion and cytotoxicity in NK cells. These novel findings suggest that LA may inhibit NK cell function via the cAMP signaling pathway.

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.

Identification and characterization of RHOA-interacting proteins in bovine spermatozoa.

In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro.

The role of protein kinase A and A-kinase anchoring proteins in modulating T-cell activation: progress and future directions.

Protein kinase A (PKA) is a broad-specificity serine/threonine protein kinase whose spatial and temporal regulation is maintained through interactions with A-kinase anchoring proteins (AKAPs). Subcellular localization of AKAPs through unique targeting domains provides a mechanism by which PKA can respond to localized microdomains of cyclic AMP (cAMP) and phosphorylate nearby substrates. For nearly 40 years, cAMP has been known to be a potent modulator of the immune system. cAMP levels are regulated by G-protein-coupled receptors, adenylyl cyclases (AC), and phosphodiesterases (PDEs). This review discusses recent progress made in the discovery of PKA substrates in T lymphocytes and in the identification of AKAPs in T lymphocytes. Because PKA is activated by cAMP, generation and maintenance of cAMP in T cells is also discussed. These findings are framed in the context of understanding the complexity of cAMP and, thus, PKA signaling and are intended to provide the reader with an overview of current literature, as well as an awareness of questions and concerns to consider.

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa.

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.

The role of protein kinase A anchoring via the RII alpha regulatory subunit in the murine immune system.

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.

A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines.

The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.