Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions

Lobban, Margaret; Shakur, Yasmin; Beattie, James; Houslay, Miles D.

doi:10.1042/bj3040399

Cited by 80 publications

(66 citation statements)

References 36 publications

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“…After lipid raft separation of CD28-stimulated primary T cells, all fractions were analyzed for PDE activity as described previously (28,31). Assays were carried out at 1 M cAMP substrate concentration in duplicate in the presence or absence of the PDE4-selective inhibitor rolipram (10 M), and thereafter, the fraction of rolipram-dependent PDE4 activity was calculated as a reflection of PDE4 activity.…”

Section: Methodsmentioning

confidence: 99%

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

Bjørgo

Solheim

Abrahamsen

et al. 2010

Molecular and Cellular Biology

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Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-T-cell receptor (TCR) stimulation alone is insufficient for activation of T cells, and sustainable T-cell immune responses require a second signal in addition to the TCR-mediated signal. The second signal is typically elicited by ligands B7-1 or B7-2 on antigen-presenting cells engaging the coreceptor CD28 to prevent anergy and apoptosis and enhancing interleukin-2 (IL-2) production and clonal expansion (4). Although CD28 plays a central role in T-cell activation in vivo (5), relatively little is known about the molecular basis for the increased efficacy of T-cell activation upon TCR and CD28 costimulation. Involvement of Lck, Itk, phosphatidylinositol 3-kinase (PI3K), SLP-76, Vav-1, and phospholipase C-␥ (PLC-␥) has, however, been reported (43). CD28-mediated signals are transmitted via a short intracellular stretch in the receptor containing a conserved YMNM motif (32). Phosphorylation of Tyr173 in this motif by Lck and Fyn following CD28 ligation is key to efficient signal transduction (41), generating a binding site for the SH2 domain of the p85 regulatory subunit of PI3K (37, 40). CD28 may also contribute to TCR-dependent PI3K activity without recruiting PI3K directly (18). Whether engagement of CD28 alone can also induce PI3K activity has been a matter of controversy. However, recent reports confirming phosphorylation of the protein kinase B (PKB) substrate glycogen synthase kinase 3 (GSK3) upon CD28 ligation has demonstrated that this is indeed the case (6, 15). In addition, CD28 can recruit growth factor receptor-bound protein 2 (Grb2), and such association of Grb2 occurs via the phosphorylated YMNM motif as well as via the C-terminal PXXP motif (22,35). The PXXP motif also binds and regulates Src family kinases (SFKs) (21, 47), and knock-in mice mutated in this motif were recently reported to have impaired IL-2 secretion (16).Ligation of the TCR induces cyclic AMP (cAMP) production (27). However, the significance of this observation is still not fully understood, as it is well established that cAMP potently inhibits T-cell function and proliferation (2,45,46,50). The spatiotemporal dynamics of the activation-induced cAMP gradient also are not completely appreciated. We have previously shown that cAMP is rapidly produced in lipid rafts following engagement of the TCR in primary T cells (3). This activates a pool of PKA type I targeted to rafts by association with the anchoring protein Ezrin, forming part of a supramolecular complex where Ezrin, EBP50, and PAG provide a scaffold that is able to coordinate PKA phosphorylation and activation of Csk, thereby inhibiting T-cell activation (44, 50). In addition, we have demonstrated that CD3/CD28 costimulation leads to recruitment of type 4 phosphodiesterase (PDE4) isoforms to rafts, resulting in degradation of the TCR-induced cAMP pool (3). Thus, we envisage that TCR-induced cAMP production constitutes a negative feedback loop capable of abrogating T-cell activation ...

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Section: Methodsmentioning

confidence: 99%

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

Bjørgo

Solheim

Abrahamsen

et al. 2010

Molecular and Cellular Biology

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“…The reason for these conflicting results is unclear. Our antibodies did not detect the PDE4B 64-kDa species described by Lobban et al (1994), which according to the authors corresponds to the DPD form (Colicelli et al, 1989). It should be pointed out that DPD is a truncated cDNA, encoding only a portion of PDE4B1 open reading frame.…”

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confidence: 50%

Characterization of the Rolipram-Sensitive, Cyclic AMP-Specific Phosphodiesterases: Identification and Differential Expression of Immunologically Distinct Forms in the Rat Brain

et al. 1998

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To determine the properties of the cAMP-specific, rolipramsensitive phosphodiesterases (cAMP-PDEs) that are expressed in different organs, monoclonal and polyclonal antibodies were raised against different epitopes present in the cAMP-PDE sequences. Of the several antibodies generated against peptides and fusion proteins, one monoclonal and four polyclonal antibodies recognized both the native cAMP-PDEs as well as the denatured proteins on Western immunoblot analysis. An immunoprecipitation assay demonstrated that these antibodies recognized the recombinant rat PDE4A, PDE4B, and PDE4D proteins with different avidity. The polyclonal antibody K118 and the monoclonal M3S1 were most specific for rat PDE4B and PDE4D forms, respectively, whereas the AC55 antiserum displayed the highest affinity for PDE4A forms. This selectivity was confirmed by Western blot analysis using recombinant rat PDE4A, PDE4B, and PDE4D proteins expressed in a heterologous system. These antibodies were used to characterize the cAMP-PDEs expressed in the rat brain. An immunoblot of extract of cortex and cerebellum demonstrated that at least seven different polypeptides specifically cross-reacted with the different antibodies, indicating that multiple cAMP-PDEs are expressed in this tissue. On the basis of cross-reactivity with PDE4D but not PDE4A or PDE4B antibodies, 93-and 105-kDa PDE4D species were detected in the cortex and cerebellum extract. These forms are different from the 68-kDa PDE4D form expressed in endocrine cells after hormonal stimulation. Although the 93-kDa form was recovered in both the soluble and particulate fractions, the 105-kDa polypeptide was mostly particulate in the cortex and cerebellum extracts. PDE4B forms of 90 -87 kDa were recovered in both soluble and particulate compartments of the brain extract. These forms were different from the previously identified PDE4A variants of 110 and 75 kDa. These data demonstrate that the presence of multiple cAMP-PDE genes is translated into cAMP-PDE proteins of different sizes and distinct immunological properties and that multiple variants derived from these cAMP-PDE genes are expressed in different regions of the brain and different subcellular compartments. These immunological tools will be useful to identify different cAMP-PDE forms expressed in organs targeted for pharmacological intervention with PDE4 inhibitors.The high affinity, cAMP-specific phosphodiesterases [type 4 according to the nomenclature proposed by Beavo et al. (1994)] are a class of enzymes with similar kinetic properties that are inhibited by the antidepressant rolipram and structurally related compounds. Although the presence of these forms has long been recognized, their distinctive properties are becoming evident only recently (Conti et al., 1995b). Early attempts to purify these forms have been hampered by their low abundance and instability (Conti and Swinnen, 1990). The molecular mass attributed to this group of enzymes ranges between 29 and 89 kDa (Conti and Swinnen, 1990). The definition of th...

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“…Homogenates from cultured cell lines or rat brain were prepared as described previously [24,30]. These homogenates were then fractionated as follows.…”

Section: Preparation and Fractionation Of Tissue Homogenatesmentioning

confidence: 99%

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene

Bolger

Erdoğan

Jones

et al. 1997

Biochemical Journal

174

229

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We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3h,5h-cyclic nucleotide phosphodiesterases (type IV PDEs ; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5h-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines

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Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions

Cited by 80 publications

References 36 publications

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

Characterization of the Rolipram-Sensitive, Cyclic AMP-Specific Phosphodiesterases: Identification and Differential Expression of Immunologically Distinct Forms in the Rat Brain

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene

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