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Biomedical subjects

M D Houslay

Publications and source records attributed to M D Houslay.

At least 19 recordsLinked to original sources
PubMed2006

Phosphodiesterase-4 gates the ability of protein kinase A to phosphorylate G-protein receptor kinase-2 and influence its translocation.

Challenge of the beta(2)Ar (beta(2)-adrenergic receptor) with isoprenaline in HEK-293beta(2) cells (human embryonic kidney cells stably overexpressing a FLAG- and green fluorescent protein-tagged beta(2)Ar) results in the PKA (cAMP-dependent protein kinase) phosphorylation of GRK2 (G-protein receptor kinase-2). This response was enhanced when PDE4 (phosphodiesterase-4) activity was attenuated using either rolipram, a PDE4-selective inhibitor, or with siRNA (small interfering RNA) knockdown of both PDE4B and PDE4D. Rolipram also facilitated GRK2 recruitment to the membrane and phosphorylation of the beta(2)Ar by GRK2 in response to isoprenaline challenge of cells. In resting cells, rolipram treatment alone is sufficient to promote PKA phosphorylation of GRK2, with consequential effects on GRK2 translocation and GRK2 phosphorylation of the beta(2)Ar. Similar effects are observed in cardiac myocytes. We propose that PDE4 activity protects GRK2 from inappropriate phosphorylation by PKA in resting cells that might have occurred through fluctuations in basal cAMP levels. Thus PDE4 gates the action of PKA to phosphorylate GRK2.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

cAMP phosphodiesterase-4A1 (PDE4A1) has provided the paradigm for the intracellular targeting of phosphodiesterases, a process that underpins compartmentalized cAMP signalling.

Specificity of cAMP signalling pathways has shown that the intracellular targeting of the individual components confers a three-dimensional context to the signalling paradigms in which they can exquisitely control the specificity of the outcome of the signal. Pivotal to this paradigm is degradation of cAMP by sequestered PDEs (phosphodiesterases). cAMP rapidly diffuses within cells and, without the action of spatially confined PDE populations, cAMP gradients could not be formed and shaped within cells so as to regulate targeted effector proteins. Of particular importance in regulating compartmentalized cAMP signalling are isoforms of the PDE4 family, which are individually defined by unique N-terminal regions. We have developed and pioneered the concept that a major function of this N-terminal region is to confer intracellular targeting of particular PDE4 isoforms on specific signalling complexes and intracellular locations. The paradigm for this concept developed from our original studies on the PDE4A1 (RD1) isoform. The N-terminal region unique to PDE4A1 consists of two well-defined helical regions separated by a mobile hinge region. Helix-2 provides the core membrane-insertion module, with helix-1 facilitating membrane association and fidelity of targeting in living cells. The irreversible, Ca(2+)-dependent insertion of the N-terminal region of PDE4A1 into membranes provides 'long-term' memory of cell activation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Compartmentalized cAMP signalling regulates vasopressin-mediated water reabsorption by controlling aquaporin-2.

The cAMP/PKA (protein kinase A) signalling pathway is activated by a plethora of stimuli. To facilitate the specificity of a cellular response, signal transduction complexes are formed and segregated to discrete sites (compartmentalization). cAMP/PKA signalling compartments are maintained by AKAPs (A-kinase anchoring proteins) which bind PKA and other signalling proteins, and by PDEs (phosphodiesterases). The latter hydrolyse cAMP and thus limit its diffusion and terminate PKA activity. An example of a cAMP-dependent process requiring compartmentalization of cAMP/PKA signals is arginine-vasopressin-regulated water reabsorption in renal principal cells. A detailed understanding of the protein interactions within a signal transduction complex offers the possibility to design agents influencing PKA binding to a specific AKAP, the targeting of an AKAP or the interactions of AKAPs with other signalling molecules. The ability to specifically modulate selected branches of a signal transduction pathway would greatly advance basic research, and may lead to new drugs suitable for the treatment of diseases caused by dysregulation of anchored PKA signalling (e.g. renal and cardiovascular diseases).

Animals↗
PubMed2005

Beta-arrestin-recruited phosphodiesterase-4 desensitizes the AKAP79/PKA-mediated switching of beta2-adrenoceptor signalling to activation of ERK.

Using combined dominant-negative and siRNA (small interfering RNA)-mediated knockdown strategies, the functional importance of specific PDE4 (phosphodiesterase-4) isoforms in modifying signalling through the beta2-AR (beta2-adrenoceptor) has been uncovered. The PDE4D5 isoform preferentially interacts with the signalling scaffold protein beta-arrestin and is thereby recruited to the beta2-AR upon agonist challenge. Delivery of an active PDE to the site of cAMP synthesis at the plasma membrane specifically attenuates the activity of a pool of PKA (protein kinase A) that is tethered to the beta2-AR via AKAP79 (A-kinase anchoring protein 79). The specific functional role of this anchored PKA is to phosphorylate the beta2-AR and allow it to switch its coupling with G(i) and thereby activation of ERK (extracellular-signal-regulated kinase). Our studies uncover a novel facet of the regulation of beta2-AR signalling by showing that beta-arrestin-recruited PDE4 provides the means of desensitizing the agonist-dependent coupling of beta2-AR with G(i) and its consequential activation of ERK.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

The role of ERK2 docking and phosphorylation of PDE4 cAMP phosphodiesterase isoforms in mediating cross-talk between the cAMP and ERK signalling pathways.

PDE4 cAMP phosphodiesterases are widely expressed enzymes that serve as major regulators of cAMP signalling in cells. They provide targets for therapeutics having anti-inflammatory and cognitive-enhancing properties. ERK2 (extracellular-signal-regulated kinase 2) interacts with the PDE4 catalytic unit by binding to a KIM (kinase interaction motif) docking site located on an exposed beta-hairpin loop and an FQF (Phe-Gln-Phe) specificity site located on an exposed alpha-helix. These flank a site that allows phosphorylation by ERK, the functional outcome of which is orchestrated by the N-terminal UCR1/2 (upstream conserved region 1 and 2) modules. The three classes of PDE4 isoforms differ in these regulatory modules, allowing phosphorylation by ERK to lead to either inhibition or activation. ERK inhibition of long isoforms is regulated by a unique feedback control whereby elevated cAMP levels cause PKA (protein kinase A) to phosphorylate UCR1 and ablate the inhibitory action of ERK. PDE4 isoforms can also be found in complex with beta-arrestins where they provide a novel part of the cellular desensitization mechanism to receptor-mediated cAMP signalling. Stimulation of the beta(2)-adrenoceptor recruits beta-arrestins with bound PDE4, delivering an enzyme capable of degrading cAMP at its site of synthesis at the plasma membrane. Use of dominant negative PDE4 isoforms identifies that a major role of recruited PDE4 is to regulate plasma membrane PKA activity involved in phosphorylating the beta(2)-adrenoceptor. Recruited PDE4 thus desensitizes the ability of the beta(2)-adrenoceptor to activate ERK via G(i).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1).

Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase-3 in in vitro and transfected Cos-7 cells. In addition, the treatment of PDE5A1-transfected Cos-7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase-3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase-3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase-3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild-type enzyme in transfected Cos-7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild-type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase-3. From this, we can predict that a caspase-3-mediated cleavage of the [778]DQGD[781] motif would result in removal of the C-terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Development of rolipram-sensitive, cyclic AMP phosphodiesterase (PDE4) in rat primary neuronal cultures.

The development of PDE4 was examined in primary neuronal cultures of rat cerebral cortex. Three days after culturing, neurons exhibited relatively low PDE4 activity (i.e., rolipram-sensitive PDE activity). It gradually increased over time, approximately doubling by day 12. The increase in activity was accompanied by an increase in the expression of the PDE4A variants, PDE4A5 and PDE4A1, as well as of the synaptic marker protein synapsin I. There was a strong correlation between the expression of the PDE4A variants with that of synapsin I, which suggests that as neurons develop and signal transduction increases there is a regulated increase in PDE4 expression and activity. Consistent with this interpretation, it was found that treatment with the sodium channel blocker tetrodotoxin, which inhibits depolarization-induced neurotransmitter release, reduced the expression of the PDE4A variants. These data demonstrate the developmental regulation of PDE4 in neurons and offer a manner by which the association of PDE4 variants with particular signal transduction pathways may be studied in vitro.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent inhibition of IL-5 from human T lymphocytes is not mediated by the cAMP-dependent protein kinase A.

IL-5 is implicated in the pathogenesis of asthma and is predominantly released from T lymphocytes of the Th2 phenotype. In anti-CD3 plus anti-CD28-stimulated PBMC, albuterol, isoproterenol, rolipram, PGE2, forskolin, cholera toxin, and the cAMP analog, 8-bromoadenosine cAMP (8-Br-cAMP) all inhibited the release of IL-5 and lymphocyte proliferation. Although all of the above compounds share the ability to increase intracellular cAMP levels and activate protein kinase (PK) A, the PKA inhibitor H-89 failed to ablate the inhibition of IL-5 production mediated by 8-Br-cAMP, rolipram, forskolin, or PGE2. Similarly, H-89 had no effect on the cAMP-mediated inhibition of lymphocyte proliferation. Significantly, these observations occurred at a concentration of H-89 (3 microM) that inhibited both PKA activity and CREB phosphorylation in intact cells. Additional studies showed that the PKA inhibitors H-8, 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphorothioate Rp isomer, and a myristolated PKA inhibitor peptide also failed to block the 8-Br-cAMP-mediated inhibition of IL-5 release from PBMC. Likewise, a role for PKG was considered unlikely because both activators and inhibitors of this enzyme had no effect on IL-5 release. Western blotting identified Rap1, a downstream target of the cAMP-binding proteins, exchange protein directly activated by cAMP/cAMP-guanine nucleotide exchange factors 1 and 2, in PBMC. However, Rap1 activation assays revealed that this pathway is also unlikely to be involved in the cAMP-mediated inhibition of IL-5. Taken together, these results indicate that cAMP-elevating agents inhibit IL-5 release from PBMC by a novel cAMP-dependent mechanism that does not involve the activation of PKA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Discriminative stimulus effects of the type-4 phosphodiesterase inhibitor rolipram in rats.

RATIONALE: Rolipram, an inhibitor of cyclic AMP phosphodiesterase (PDE4) produces discriminative stimulus effects in rats. These effects may be related to a wide range of central nervous system effects described previously. OBJECTIVE: The purposes of the present study were to: (i) assess the specificity of the discriminative stimulus effects of rolipram; (ii) examine the role of beta adrenergic receptors; (iii) assess the effects of imipramine and nisoxetine; and (iv) determine whether SKF 38393, a compound which also increases cAMP levels, substitutes for rolipram. METHODS: Rats were trained to discriminate 0.1 mg/kg rolipram from its vehicle in a two-lever task. Following discrimination training, substitution and antagonism tests were carried out. RESULTS: In generalization tests, the PDE4 inhibitors ICI 63,197 and Ro 20-1724 substituted for rolipram in a dose-dependent manner (substitution at 0.3 mg/kg and 3 mg/kg, respectively). The selective inhibitors of PDE1, PDE2, and PDE5/6 did not substitute for rolipram; however, a dose of 10 mg/kg of the PDE3 inhibitor milrinone did substitute. The beta adrenergic agonists clenbuterol and dobutamine at least partially substituted for rolipram (0.1 mg/kg and 18 mg/kg, respectively). By contrast, the D1 dopaminergic agonist SKF 38393 and the monoamine uptake inhibitors imipramine and nisoxetine were ineffective (at doses up to 3, 10, and 10 mg/kg, respectively). CONCLUSIONS: The present results indicate that the discriminative stimulus effects of rolipram are related to the inhibition of the hydrolytic activity of PDE4. Generalized increases in cyclic nucleotides do not appear to be sufficient for producing rolipram-like effects. It appears that a mechanism involving beta adrenergic receptors may contribute to the effects of rolipram, consistent with previous neuropharmacological data. Finally, the discriminative stimulus effects of rolipram appear to be unrelated to its antidepressant-like effect, but may provide a surrogate marker for central nervous system-related side effects of PDE4 inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5.

A strategy of mutagenesis followed by yeast two-hybrid assay was used to determine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodiesterase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-repeats 5-7, inclusively, of RACK1 contained the major site for interaction with PDE4D5. A reverse two-hybrid screen focusing on WD-repeats 5-7 of RACK1 isolated 11 single amino acid mutations from within this region that blocked the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathione-S-transferase (GST)-RACK1 and mammalian cell-expressed PDE4D5. A model of RACK1 structure, based on the structural similarity of RACK1 to other beta-propeller WD-repeat proteins, indicated that the majority of the amino acids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interaction with the unique amino-terminal region of PDE4D5.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

The novel long PDE4A10 cyclic AMP phosphodiesterase shows a pattern of expression within brain that is distinct from the long PDE4A5 and short PDE4A1 isoforms.

In situ hybridisation methods were used to map the distribution of the novel long PDE4A10 isoform in the brain. PDE4A10 distribution was compared to that of the long PDE4A5 isoform and the short PDE4A1 isoform using probes specific for unique sequences within each of these isoforms. Coronal sections of the brain, taken at the level of the olfactory bulb, prefrontal cortex, striatum, thalamus, hippocampus and cerebellum, were analysed. Strongest expression of PDE4A isoforms was found in the olfactory bulb granular layer with high signals also in the piriform cortex, the dentate gyrus and the CA1 and CA2 pyramidal cells. For the two long forms, level general staining was noted throughout the striatum, thalamus and hippocampus but no signal was evident in the cerebellum. The long PDE4A10 and PDE4A5 isoforms localised to essentially the same regions throughout the brain, although PDE4A10 was uniquely expressed in the major island of Calleja. A signal for the short PDE4A1 isoform was found in regions in which the two long isoforms were both expressed, with the exception of the medial nucleus of the amygdala where weak signals for PDE4A5 and PDE4A10 were detected but PDE4A1 was absent. Uniquely, strong signals for PDE4A1 were detected in the glomerular layer of the olfactory bulb, the CA3 pyramidal cell region and the cerebellum; areas where signals for the two long forms were not evident. PDE4A transcripts for both PDE4A5 and PDE4A10 were not apparent in the brain stem and those for PDE4A1 were low. PDE4A isoforms are present in several key areas of the brain and therefore present valid targets for therapeutic interventions. Whilst the two long PDE4A isoforms show a remarkably similar distribution, in at least three regions there is clear segregation between their pattern of expression and that of the PDE4A1 short form. This identifies differential regulation of the expression of PDE4A long and short isoforms. We suggest that specific PDE4A isoforms may have distinct functional roles in the brain, indicating that PDE4A isoform-selective inhibitors may have specific therapeutic and pharmacologic properties.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats.

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Molecular cloning, genomic positioning, promoter identification, and characterization of the novel cyclic amp-specific phosphodiesterase PDE4A10.

We describe the cloning and expression of HSPDE4A10, a novel long form splice variant of the human cAMP phosphodiesterase PDE4A gene. The 825 amino acid HSPDE4A10 contains a unique N terminus of 46 amino acids encoded by a unique 5' exon. Exon-1(4A10) lies approximately 11 kilobase pairs (kb) downstream of exon-1(4A4) and approximately 13.5 kb upstream of the PDE4A common exon 2. We identify a rat PDE4A10 ortholog and reveal a murine ortholog by nucleotide sequence database searching. PDE4A10 transcripts were detected in various human cell lines and tissues. The 5' sequence flanking exon-1(4A10) exhibited promoter activity with the minimal functional promoter region being highly conserved in the corresponding mouse genomic sequence. Transient expression of the engineered human PDE4A10 open reading frame in COS7 cells allowed detection of a 121-kDa protein in both soluble and particulate fractions. PDE4A10 was localized primarily to the perinuclear region of COS7 cells. Soluble and particulate forms exhibited similar K(m) values for cAMP hydrolysis (3-4 microM) and IC(50) values for inhibition by rolipram (50 nM) but the V(max) value of the soluble form was approximately 3-fold greater than that of the particulate form. At 55 degrees C, soluble HSPDE4A10 was more thermostable (T(0.5) = 11 min) than the particulate enzyme (T(0.5) = 5 min). HSPDE4A10 and HSPDE4A4B are shown here to be similar in size and exhibit similar maximal activities but differ with respect to sensitivity to inhibition by rolipram, thermostability, interaction with the SRC homology 3 domain of LYN, an SRC family tyrosyl kinase, and subcellular localization. We suggest that the unique N-terminal regions of PDE4A isoforms confer distinct properties upon them.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Phorbol 12-myristate 13-acetate triggers the protein kinase A-mediated phosphorylation and activation of the PDE4D5 cAMP phosphodiesterase in human aortic smooth muscle cells through a route involving extracellular signal regulated kinase (ERK).

Phosphodiesterase 4D5 is the sole PDE4D cAMP phosphodiesterase isoform expressed in human aortic smooth muscle cells (HASMC). Phorbol 12-myristate 13-acetate (PMA) challenge of HASMC rapidly activated PDE4D5 through a process ablated by the mitogen-activated protein kinase kinase inhibitor PD98059. PMA elicited an inhibitory effect on PDE4D5 activity in HASMC treated with the cyclooxygenase (COX) inhibitor indomethacin, the COX-2 selective inhibitor NS-398, the phospholipase A(2) inhibitor quinacrine, and the cAMP-dependent protein kinase A (PKA) inhibitor H89. PMA challenge of COS-1 cells elicited the rapid inhibition and phosphorylation of both recombinant and endogenous PDE4D5 in a manner ablated by PD98059 and not seen in S651A mutant PDE4D5. PMA promoted the generation of PGE(2) in the medium of HASMC and caused activation of both extracellular signal-regulated kinase (ERK) and PKA through a process ablated by indomethacin, NS-398, quinacrine, and PD98059. Exogenous prostaglandin (PG) E(2) increased cAMP levels and activated PKA in HASMC. COX-2 was expressed in HASMC but not in COS-1 cells. Forskolin challenge of COS-1 cells activated PDE4D5 by causing the PKA-mediated phosphorylation of Ser126 as detected using a novel phosphospecific antiserum. PMA challenge of HASMC elicited phosphorylation of the stimulatory PKA-specific phosphorylation site, Ser126 in PDE4D5 in a manner ablated by PD98059, indomethacin, and H89. We propose that, in HASMC, PMA activates PDE4D5 through an ERK-controlled autocrine mechanism. This involves PGE(2) generation, which causes activation of adenylyl cyclase, allowing PKA to elicit net activation of PDE4D5 by phosphorylation at Ser126.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum.

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3. Consequences for altered intracellular distribution.

The unique N-terminal region of the cAMP-specific phosphodiesterase PDE4A5, which confers an ability to bind to certain protein SH3 domains, is cleaved during apoptosis in both Rat-1 fibroblasts and PC12 cells. Cleavage was abolished by the caspase-3-selective inhibitor, z-DEVD-CHO but not the caspase-1 selective inhibitor, z-YVAD-CHO. Caspase-3 treatment of PDE4A5, expressed either transiently in COS cells or generated in vitro by coupled transcription translation, generated a similar cleavage product of 100 kDa compared with the native 110-kDa PDE4A5. This product could be detected immunochemically with an antibody raised to a C-terminal PDE4A5 peptide but not an antibody raised to the N terminus of PDE4A5, indicating that caspase-3 caused N-terminal cleavage of PDE4A5. Deletion of the putative caspase-3 cleavage site, (69)DAVD(72), in PDE4A5, or generation of either the D72A or the D69A mutants, ablated the ability of caspase-3 to cause cleavage. The N-terminal truncate PDE4A5-DeltaP3 was engineered to mimic the caspase-cleaved product of PDE4A5. This showed altered catalytic activity and, unlike PDE4A5, was unable to interact with the SH3 domain of the tyrosyl kinase, LYN. Although both PDE4A5 and PDE4A5-DeltaP3 were localized at cell cortical regions (ruffles), the distinct perinuclear association noted for both PDE4A5 and LYN was not seen for PDE4A5-DeltaP3. Staurosporine-induced apoptosis caused a marked redistribution of PDE4A5 but not PDE4A8 in stably transfected Rat-1 cells. The PDE4-selective inhibitor, rolipram together with the adenylyl cyclase activator forskolin, caused a synergistic increase in the apoptosis of Rat-1 cells. Overexpression of PDE4A5 in Rat-1 cells protected against staurosporine-induced apoptosis in contrast to overexpression of PDE4A8, which potentiated apoptosis. PDE4A5 may be the sole PDE4 family member to provide a substrate for caspase-3 cleavage and this action serves to remove the SH3 binding domain that is unique to this isoform within the PDE4A family and to alter its intracellular targeting.

3',5'-Cyclic-AMP Phosphodiesterases↗