Absence Of cAMP Research Articles

Pterosin B has multiple targets in gluconeogenic programs, including coenzyme Q in RORα–SRC2 signaling

Hepatic gluconeogenic programs are regulated by a variety of signaling cascades. Glucagon–cAMP signaling is the main initiator of the gluconeogenic programs, including glucose-6-phosphatase catalytic subunit (G6pc) gene expression. Pterosin B, an ingredient in Pteridium aquilinum, inhibits salt-inducible kinase 3 signaling that represses cAMP-response element-binding protein regulated transcription coactivator 2, an inducer of gluconeogenic programs. As the results, pterosin B promotes G6pc expression even in the absence of cAMP. In this work, however, we noticed that once cAMP signaling was initiated, pterosin B became a strong repressor of G6pc expression. The search for associated transcription factors for pterosin B actions revealed that retinoic acid receptor-related orphan receptor alpha–steroid receptor coactivator 2 (RORα–SRC2) complex on the G6pc promoter was the target. Meanwhile, pterosin B impaired the oxidation–reduction cycle of coenzyme Q in mitochondrial oxidative phosphorylation (OXPHOS); and antimycin A, an inhibitor of coenzyme Q: cytochrome c–oxidoreductase (termed mitochondrial complex III), also mimicked pterosin B actions on RORα–SRC2 signaling. Although other respiratory toxins (rotenone and oligomycin) also suppressed G6pc expression accompanied by lowered ATP levels following the activation of AMP-activated kinase, minimal or no effect of these other toxins on RORα–SRC2 activity was observed. These results suggested that individual components in OXPHOS differentially linked to different transcriptional machineries for hepatic gluconeogenic programs, and the RORα–SRC2 complex acted as a sensor for oxidation–reduction cycle of coenzyme Q and regulated G6Pc expression. This was a site disrupted by pterosin B in gluconeogenic programs.

Directed Evolution of the Escherichia coli cAMP Receptor Protein at the cAMP Pocket

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling

The second messenger molecule cAMP regulates the activation phase of the cAMP signaling pathway through high-affinity interactions with the cytosolic cAMP receptor, the protein kinase A regulatory subunit (PKAR). Phosphodiesterases (PDEs) are enzymes responsible for catalyzing hydrolysis of cAMP to 5′ AMP. It was recently shown that PDEs interact with PKAR to initiate the termination phase of the cAMP signaling pathway. While the steps in the activation phase are well understood, steps in the termination pathway are unknown. Specifically, the binding and allosteric networks that regulate the dynamic interplay between PKAR, PDE, and cAMP are unclear. In this study, PKAR and PDE from Dictyostelium discoideum (RD and RegA, respectively) were used as a model system to monitor complex formation in the presence and absence of cAMP. Amide hydrogen/deuterium exchange mass spectrometry was used to monitor slow conformational transitions in RD, using disordered regions as conformational probes. Our results reveal that RD regulates its interactions with cAMP and RegA at distinct loci by undergoing slow conformational transitions between two metastable states. In the presence of cAMP, RD and RegA form a stable ternary complex, while in the absence of cAMP they maintain transient interactions. RegA and cAMP each bind at orthogonal sites on RD with resultant contrasting effects on its dynamics through parallel allosteric relays at multiple important loci. RD thus serves as an integrative node in cAMP termination by coordinating multiple allosteric relays and governing the output signal response.

Characterization of a novel mutation in fibrolamellar hepatocellular carcinoma.

284 Background: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a subtype of HCC occurring in children and young adults in the absence of underlying liver disease. Currently, there is no effective therapy for unresectable or metastatic FL-HCC. Recent genomic analysis identified a consistent mutation in FL-HCC involving a deletion on chromosome 19 resulting in a chimeric transcript containing the 5’-region of a heat shock protein (DNAJB1) fused to the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant protein and its effects on PKA activity in human FL-HCC. Methods: We prepared tissue lysates from four snap-frozen FL-HCC samples with paired, non-tumor liver as well as adult HCCs. PKA activity was determined via a radioactive kinase assay in the presence and absence of cAMP, a PKA activator. RNA was extracted using TRIZOL, and used for qRT-PCR. Triple immuno-fluorescent labeling was performed using antibodies to PRKACA, PKA RIIα, and a nuclear marker, DRAQ5. Results: We found that expression of the chimeric transcript was increased 40-fold in FL-HCC compared to normal liver, and was significantly higher than that of wild-type DNAJB1 and PRKACA in FL-HCC. The corresponding mutant protein was highly expressed in the tumors and was unique to FL-HCC. Basal PKA activities from freshly lysed tumors and paired livers were not significantly different, but cAMP-stimulated PKA activity was significantly higher in FL-HCC tumors when compared to normal liver. Using multi-color immuno-fluorescence we detected mutant DNAJB1-PRKACA within the nucleus, while wild-type PRKACA localizes to the cytoplasm. Conclusions: The expression of DNAJB1-PRKACA is unique to FL-HCC and not found in classic forms of HCC. Markedly elevated expression of the mutant transcript is indicative of dysregulated DNAJB1 promoter activity. PKA activity in FL-HCCs remains cAMP-dependent, but is greatly enhanced in the presence of cAMP; this could reflect an altered intrinsic activity of the mutant protein and/or elevated expression. Further, the mutant protein showed aberrant localization to the nucleus. These findings suggest that DNAJB1-PRKACA in FL-HCC leads to over-activation of PKA, which may contribute to tumor development.

Computational Studies on the cAMP Modulation of the HCN2 Channel

Hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels play a fundamental role in electric signaling in nerves, muscles, and synapses; however, their ligand gating mechanism is not well understood. Through collaboration between experimental and computational methods, this study brings insight into the mechanism of cyclic adenosine monophosphate (cAMP) modulation of the HCN2 channel (residues 443-636) by exploring the monomer and tetramer dynamics in the apo and holo states. We applied all-atom and coarse-grained molecular dynamics on molecular systems containing HCN2 with and without cAMP. Starting from the holo structure resolved by X-ray crystallography, our simulations guide the protein into its apo state. Along this pathway, we observed unfolding of the C'-helix and loss of contact between A’ and B’ helices in the C-linker and the cyclic nucleotide binding domain (CNBD). In addition, by using tmFRET distances as restraints, we were able to capture structural changes in the CNBD. Our results corroborate recent experimental studies showing the outward movement of the C-helix in the absence of cAMP.

Revisiting the mechanism of activation of cyclic AMP receptor protein (CRP) by cAMP in Escherichia coli: Lessons from a subunit-crosslinked form of CRP

Cyclic AMP receptor protein (CRP), the global transcription regulator in prokaryotes, is active only as a cAMP–CRP complex. Binding of cAMP changes the conformation of CRP, transforming it from a transcriptionally ‘inactive’ to an ‘active’ molecule. These conformers are also characterized by distinct biochemical properties including the ability to form an S–S crosslink between the C178 residues of its two monomeric subunits. We studied a CRP variant (CRPcl), in which the subunits are crosslinked. We demonstrate that CRPcl can activate transcription even in the absence of cAMP. Implications of these results for the crystallographically-determined structure of cAMP–CRP are discussed.

Paralogous cAMP receptor proteins in Mycobacterium smegmatis show biochemical and functional divergence.

The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78% identical sequences and characterize them biochemically and functionally. The two proteins (MSMEG_0539 and MSMEG_6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg_3781 gene. MSMEG_6189 binds to the CRP site readily in the absence of cAMP, while MSMEG_0539 binds in the presence of cAMP, albeit weakly. msmeg_6189 appears to be an essential gene, while the Δmsmeg_0539 strain was readily obtained. Using promoter-reporter constructs, we show that msmeg_3781 is regulated by CRP binding, and its transcription is repressed by MSMEG_6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism.

CAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells.

Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8.

A Mechanism for the Auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Opening and Its Relief by cAMP

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.

Role of arginine 209 in the conformational transition of the protein kinase A regulatory subunit RIα A-domain

Using the combination of molecular dynamics (MD) simulations and geometric clustering we analyzed the role of arginine at 209 position in the transition of protein kinase A Iα (PKA Iα) regulatory subunit A-domain from H- to B-conformation and stabilization of the latter. The mechanism underlying the role of the residue at position 209 in the realization of B-conformation includes: (1) possibility to bind the ligand tightly (if transition happens in the presence of cAMP), (2) capability to hold β2β3-loop in the correct conformation, (3) tendency of residue at 209 position to stabilize B-conformation in the absence and in presence of the ligand. In terms of the effect produced on transition of A-domain from H- to B-conformation in the presence of cAMP, mutational substitutions for R209 can be arranged in the following order: Glu(Gly)>Lys>Ile. In the absence of cAMP the order is different Lys>Gly>Glu>Ile. Thus, our results allow us to presume that the role of arginine at 209 position can be important though not crucial.

Different Effects of Alkaline Phosphatase on HCN4 Channels in CHO Versus HEK Cells

Hyperpolarization-activated, cyclic nucleotide-sensitive type 4 (HCN4) channels are regulated by cAMP, which can bind directly to a conserved cyclic nucleotide binding domain (CNBD) in the C-terminus. We previously found that the response of HCN4 channels to cAMP depends on the cellular context: cAMP binding to the CNBD shifts the midpoint activation voltage (V1/2) to more depolarized potentials when HCN4 is expressed in HEK cells, whereas cAMP has no effect on the V1/2 when HCN4 is expressed in CHO cells because the channels are already pre-activated, with a depolarized V1/2 in the absence of cAMP (Liao et al., 2012 J Gen Physiol 140(5):557). Here we have tested the hypothesis that differential phosphorylation may underlie the differences in HCN4 behavior in CHO versus HEK cells. We found that while alkaline phosphatase (AlkPhos) restored the hyperpolarized basal V1/2 of HCN4 in CHO cells, it did not restore the cAMP sensitivity of the channels. Quite different results were obtained in HEK cells, in which AlkPhos had little or no effect on the V1/2 of HCN4 in the absence of cAMP but, remarkably, was found to significantly reduce the shift in V1/2 in response to cAMP. These results indicate that dual regulation of HCN4 channels by cAMP and phosphorylation is complex and is sensitive to cellular context. And, phosphorylation alone is insufficient to account for the different behaviors of HCN4 channels in CHO versus HEK cells.

Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

Hyperpolarization-activated cyclic nucleotide–regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

PKA-independent activation of If by cAMP in mouse sinoatrial myocytes

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN4) channels produce the “funny current,” If, which contributes to spontaneous pacemaking in sinoatrial myocytes (SAMs). The C-terminus of HCN channels inhibits voltage-dependent gating, and cAMP binding relieves this “autoinhibition.” We previously showed 1) that autoinhibition in HCN4 can be relieved in the absence of cAMP in some cellular contexts and 2) that PKA is required for β adrenergic receptor (βAR) signaling to HCN4 in SAMs. Together, these results raise the possibility that native HCN channels in SAMs may be insensitive to direct activation by cAMP. Here, we examined PKA-independent activation of If by cAMP in SAMs. We observed similar robust activation of If by exogenous cAMP and Rp-cAMP (an analog than cannot activate PKA). Thus PKA-dependent βAR-to-HCN signaling does not result from cAMP insensitivity of sinoatrial HCN channels and might instead arise via PKA-dependent limitation of cAMP production and/or cAMP access to HCN channels in SAMs.

Inactivated spHCN Channel has a Decreased Binding Affinity for Camp

In response to both voltage and ligand, HCN channels play important physiological roles in the brain and the heart. HCN channels are activated by membrane hyperpolarization and the direct binding of intracellular cAMP. The spHCN channel was cloned from Sea Urchin and belongs to the HCN channel family. Different from the mammalian HCN1-4 channels, the spHCN channel exhibits strong inactivation in the absence of cAMP. Application to cAMP to WT spHCN channel abolishes the inactivation. Interestingly, a previously identified point mutation near the inner activation gate in S6, F459L, makes the channel behave just like the mammalian HCN channels with any inactivation. Taking advantage of the patch-clamp fluorometry technique, we set out to investigate the dynamic, activity-dependent cAMP binding during spHCN channel gating. Surprisingly, during channel activation, we observed a decrease in cAMP binding, which is directly opposite to the observation with the HCN2 channel. Conversely, in the spHCN/F459L mutant channel, we observed an increase in cAMP binding during channel activation, which is similar to that observed in HCN2 channel. These observations provide new insights into the intriguing communication between the voltage-dependent and ligand-dependent gating in HCN channels.

Voltage- and Camp-Dependent Gating in Heterotetrameric HCN2/4-Pacemaker Channels

HCN pacemaker channels play an important role in generating and regulating rhythmicity of special neurons and cardiac cells. They are activated by hyperpolarizing voltages and modulated by the binding of cyclic nucleotides. Four isoforms, HCN1-HCN4, have been identified. HCN2 and HCN4, expressed in cardiac sinoatrial node and ventricular cells, build functional homotetrameric and heterotetrameric channels in various heterologous cell systems. Heterotetrameric HCN2/4 channels in Xenopus oocytes show two changes compared to each of the homotetramers: the voltage of half maximum activation is shifted to more depolarized voltages and activation kinetics are faster (Zhang et al., Biochim Biophys Acta, 2009). However, little is known about the ligand dependence of these channels. Herein, we studied the differences in activation gating of HCN2/4 channels in comparison to the respective homotetramers, thereby focusing on the effect of cAMP. We monitored activation under both steady-state and non steady-state conditions in the presence and absence of cAMP, as well as the ligand-dependent activation kinetics after ligand jumps. We found (1) that in HCN2/4 the apparent affinity for cAMP was between that of the two homotetramers, whereas the Hill coefficient was lowest, (2) that cAMP accelerates voltage-induced activation in HCN2/4 only slightly (factor ∼2), resembling HCN4, whereas in HCN2 it accelerates activation in a voltage-dependent manner by a factor of up to ∼12, (3) that the activation kinetics following a cAMP jump to channels pre-activated by voltage was fastest in HCN2/4, whereas the increase of current amplitude by a concentration jump was similar to homotetramers. Our results confirm that coinjection of the HCN2 and HCN4 isoforms in Xenopus oocytes leads to heterotetrameric channels. They suggest that in native heart cells the formation of heterotetramers leads to pacemaker channels with specific characteristics, thereby fine-tuning the process of pacemaking.

The Auto-Inhibitory Role of the EPAC Hinge Helix as Mapped by NMR

The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.

Cyclic AMP regulates formation of mammary epithelial acini in vitro

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

Function analysis of conserved amino acid residues in a Mn2+-dependent protein phosphatase, Pph3, from Myxococcus xanthus

The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg(2+)- or Mn(2+)-dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal- or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound.

CAMP regulates polarization and apoptosis during mammary epithelial acini formation in vitro

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three‐dimensional (3D) culture models utilizing MCF10A mammary epithelial cells, hollow acini form due to integrin‐dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking ECM contact. Here, we report that cAMP‐dependent protein kinase A (PKA) promotes hollow acini formation via two mechanisms. First, cAMP accelerates the polarization of outer cells due to ERK/MAPK inhibition. Second, cAMP, promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed resulting in profound luminal filling. cAMP‐dependent apoptosis is accompanied by a posttrascriptional increase in the proapoptotic protein BIM, which is at least partially ERK‐independent. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cell cultures both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.This study was supported by NIH grants to K.E.M (R01 DK074398 and P01 AI53194), S.K. (DK082115), and J.D (RO1 CA126792). P.I.N. was supported by a fellowship from the German Research Society (DFG; Ne‐897/1‐1).