Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli

Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication.     

Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases

Histone deacetylase 4 (HDAC4) and its paralogs, HDAC5, -7, and -9 (all members of class IIa), possess multiple phosphorylation sites crucial for 14-3-3 binding and subsequent nuclear export. cAMP signaling stimulates nuclear import of HDAC4 and HDAC5, but the underlying mechanisms remain to be elucidated. Here we show that cAMP potentiates nuclear localization of HDAC9. Mutation of an SP motif conserved in HDAC4, -5, and -9 prevents cAMP-stimulated nuclear localization. Unexpectedly, this treatment inhibits phosphorylation at the SP motif, indicating an inverse relationship between the phosphorylation event and nuclear import. Consistent with this, leptomycin B-induced nuclear import and adrenocorticotropic hormone (ACTH) treatment result in the dephosphorylation at the motif. Moreover, the modification synergizes with phosphorylation at a nearby site, and similar kinetics was observed for both phosphorylation events during myoblast and adipocyte differentiation. These results thus unravel a previously unrecognized mechanism whereby cAMP promotes dephosphorylation and differentially regulates multisite phosphorylation and the nuclear localization of class IIa HDACs.     

A cyclic adenosine 3',5'-monophosphate-induced tyrosine phosphorylation of Syk protein tyrosine kinase in the flagella of boar spermatozoa

A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5 degrees C for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the anti-phospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+-independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa.     

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.     

Compartmentalization of PDE-4 and cAMP-dependent protein kinase in neutrophils and macrophages during phagocytosis

The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils, and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D, and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4 at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization.     

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs,an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.     

Altered expression of genes regulating cell growth,proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture

An elevation of the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing approximately 6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4, 24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppressors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.     

Role of the EHD2 Unstructured Loop in Dimerization,Protein Binding and Subcellular Localization

The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2’s homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.     

磷酸二酯酶的心血管功能调节作用

磷酸二酯酶(phosphodiesterase,PDE)是细胞内第二信使cAMP和cGMP降解的关键酶,作为药物研发的靶点受到广泛关注。近年研究发现,PDE在心肌细胞中能与β-肾上腺素受体及一些与兴奋收缩相关的蛋白形成复合物而使细胞内信号传递区室化分布,该现象可能为PDE抑制剂治疗慢性心力衰竭提供新的启示。血管平滑肌功能调节主要为血管张力和表型的调控,PDE5抑制剂舒张血管的作用已成功应用到勃起障碍的治疗。PDE4和PDE1C等在增殖的平滑肌细胞中表达量增高,单独抑制PDE的某一亚型将为治疗与平滑肌增殖有关的疾病(如肺动脉高压、血管成行术后再狭窄)提供新的途径。本文将重点阐述近年来PDE在心血管系统功能调节研究中的主要进展,以及PDE抑制剂在心血管系统疾病治疗中的应用。     

Improvement of pulmonary arterial hypertension,inflammatory response,and epithelium injury by dual activation of cAMP/cGMP pathway in a rat model of monocrotaline-induced pulmonary hypertension

Pulmonary hypertension (PH) is a life-threatening lung disease. PH with concomitant lung diseases, e.g., idiopathic pulmonary fibrosis, is associated with poor prognosis. Development of novel therapeutic vasodilators for treatment of these patients is a key imperative. We evaluated the efficacy of dual activation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using an active, small-molecule phosphodiesterase (PDE4)/PDE5 dual inhibitor (Compound A). Compound A increased both cAMP and cGMP levels in WI-38 lung fibroblasts and suppressed the expressions of type-1 collagen α1 chain and fibronectin. Additionally, compound A reduced right ventricular weight/left ventricular weight+septal weight ratio, brain natriuretic peptide expression levels in right ventricle, C─C motif chemokine ligand 2 expression levels in lung, and plasma surfactant protein D. Our data indicate that dual activation of cAMP/cGMP pathways may be a novel treatment strategy for PH.     

Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.     

Quantitative phosphoproteomics reveals novel roles of cAMP in plants

3′,5′-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3′,5′-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular “sponge” that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.     

Differential modulation of evoked and spontaneous glycine release from rat spinal cord glycinergic terminals by the cyclic AMP/protein kinase A transduction cascade

The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-clamp recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC amplitude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC amplitude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.     

Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae

A previous study identified MoRgs1 as an RGS protein that negative regulates G-protein signaling to control developmental processes such as conidiation and appressorium formation in Magnaporthe oryzae. Here, we characterized additional seven RGS and RGS-like proteins (MoRgs2 through MoRgs8). We found that MoRgs1 and MoRgs4 positively regulate surface hydrophobicity, conidiation, and mating. Indifference to MoRgs1, MoRgs4 has a role in regulating laccase and peroxidase activities. MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 are important for germ tube growth and appressorium formation. Interestingly, MoRgs7 and MoRgs8 exhibit a unique domain structure in which the RGS domain is linked to a seven-transmembrane motif, a hallmark of G-protein coupled receptors (GPCRs). We have also shown that MoRgs1 regulates mating through negative regulation of Gα MoMagB and is involved in the maintenance of cell wall integrity. While all proteins appear to be involved in the control of intracellular cAMP levels, only MoRgs1, MoRgs3, MoRgs4, and MoRgs7 are required for full virulence. Taking together, in addition to MoRgs1 functions as a prominent RGS protein in M. oryzae, MoRgs4 and other RGS and RGS-like proteins are also involved in a complex process governing asexual/sexual development, appressorium formation, and pathogenicity.     

mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module

Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA- mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.