Recombinant Expression of a Type IV, cAMP-Specific Phosphodiesterase:  Characterization and Structure−Function Studies of Deletion Mutants

Kovala, A. Thomas; Sanwal, B. D.; Ball, Eric H.

doi:10.1021/bi9613483

Cited by 52 publications

(30 citation statements)

References 47 publications

(74 reference statements)

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“…8A). Such an increase in PDE activity following deletion and/or mutation of the N terminus has been previously reported for PDE3 and PDE4 (24,25,43). Despite the dramatic increase in PDE activity, this mutant failed to enhance the production of IL-2 in response to SEE and APCs (Fig.…”

Section: Expression Of Functional Pde4b2-gfp In Jurkat T Cellssupporting

confidence: 78%

Regulation of T-Cell Activation by Phosphodiesterase 4B2 Requires Its Dynamic Redistribution during Immunological Synapse Formation

Arp

Kirchhof

Baroja

et al. 2003

Molecular and Cellular Biology

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Stimulation of T cells through their antigen receptors (TCRs) causes a transient increase in the intracellularconcentration of cyclic AMP (cAMP). However, sustained high levels of cAMP inhibit T-cell responses, suggesting that TCR signaling is coordinated with the activation of cyclic nucleotide phosphodiesterases (PDEs). The molecular basis of such a pathway is unknown. Here we show that TCR-dependent signaling activates PDE4B2 and that this enhances interleukin-2 production. Such an effect requires the regulatory N terminus of PDE4B2 and correlates with partitioning within lipid rafts, early targeting of this PDE to the immunological synapse, and subsequent accumulation in the antipodal pole of the T cell as activation proceeds.

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Section: Expression Of Functional Pde4b2-gfp In Jurkat T Cellssupporting

confidence: 78%

Regulation of T-Cell Activation by Phosphodiesterase 4B2 Requires Its Dynamic Redistribution during Immunological Synapse Formation

Arp

Kirchhof

Baroja

et al. 2003

Molecular and Cellular Biology

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“…Another study with PDE3 showed similar K m values with amino-terminal deletion mutants, but the V max values increased with larger amino-terminal deletions (40). Similarly, several studies involving PDE4 amino-terminal deletion mutants determined that these enzymes had similar K m values as did wild type PDE4, but increased specific enzyme activity (15,42). These results reveal a similar pattern of properties shared by PDE catalytic domains, and support the notion that results from our studies with PDE5 catalytic fragment may apply to those of other mammalian PDEs.…”

supporting

confidence: 85%

Expression of an Active, Monomeric Catalytic Domain of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)

Fink¹,

Francis²,

Beasley

et al. 1999

Journal of Biological Chemistry

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Phosphodiesterases (PDEs) comprise a superfamily of phosphohydrolases that degrade 3,5-cyclic nucleotides. All known mammalian PDEs are dimeric, but the functional significance of dimerization is unknown. A deletion mutant of cGMP-binding cGMP-specific PDE (PDE5), encoding the 357 carboxyl-terminal amino acids including the catalytic domain, has been generated, expressed, and purified. The K m of the catalytic fragment for cGMP (5.5 ؎ 0.51 M) compares well with those of the native bovine lung PDE5 (5.6 M) and full-length wild type recombinant PDE5 (2 ؎ 0.4 M). The catalytic fragment and full-length PDE5 have similar IC 50 values for the inhibitors 3-isobutyl-1-methylxanthine (20 M) and sildenafil (Viagra TM )(4 nM). Based on measured values for Stokes radius (29 Å) and sedimentation coefficient (2.9 S), the PDE5 catalytic fragment has a calculated molecular mass of 35 kDa, which agrees well with that predicted by amino acid content (43.3 kDa) and with that estimated using SDS-polyacrylamide gel electrophoresis (39 kDa). The combined data indicate that the recombinant PDE5 catalytic fragment is monomeric, and retains the essential catalytic features of the dimeric, full-length enzyme. Therefore, the catalytic activity of PDE5 holoenzyme requires neither interaction between the catalytic and regulatory domains nor interactions between subunits of the dimer.Intracellular levels of cyclic nucleotide are determined both by their rates of formation by adenylyl cyclases and guanylyl cyclases and their rates of breakdown by cyclic nucleotide phosphodiesterases (PDEs) 1 (1). Both the cyclases and the PDEs are highly regulated (2-8). Mammalian PDEs form a superfamily of enzymes comprised of 10 families of PDEs which differ in amino acid sequences (2, 3, 9 -11), substrate specificities, inhibitor sensitivities, modes of regulation, and tissue distribution. PDEs are chimeric proteins (12-15) which contain specific domains that provide for cyclic nucleotide hydrolysis, interaction with allosteric/regulatory molecules, autoinhibition, subcellular localization, and perhaps for other functions as well (12,16,17). For mammalian PDEs, the regulatory domains appear to reside mainly in the amino-terminal portions of the proteins. The carboxyl-terminal portion of each PDE contains a highly conserved region of ϳ250 amino acids (4), which comprises the catalytic domain.The cGMP-binding cGMP-specific PDE (PDE5 or cG-BPDE), which is the focus of this study, is abundant in lung, platelets, and vascular smooth muscle (18 -23). It is potently inhibited by sildenafil (Viagra TM ), which has proven to be therapeutically efficacious in the treatment of male erectile dysfunction (24). The PDE5 is highly specific for cGMP, both in its catalytic site, and in the two cGMP-binding allosteric sites within the aminoterminal half of the protein (14, 25). The regulation of the PDE5 catalytic activity is not well understood, although evidence has been presented that catalytic function is influenced by elements within the regulatory domain (26,27). Cycl...

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“…In the presence of UCR1, ERK phosphorylation leads to suppression of PDE4D catalytic activity; whereas in the absence of UCR1, the functional consequence of this modification is activation (22)(23)(24). On the other hand, UCR2 bears an autoinhibitory property against PDE4D catalytic activity (25)(26)(27). Given that ERK hyperactivation is common across human cancers, loss of UCR1/UCR2 caused by the genomic microdeletions will conceivably lead to PDE4D activation.…”

Section: Discussionmentioning

confidence: 99%

Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers

Lin

Ding

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Discovery of cancer genes through interrogation of genomic dosage is one of the major approaches in cancer research. In this study, we report that phosphodiesterase subtype 4D (PDE4D) gene was homozygously deleted in 198 cases of 5,569 primary solid tumors (3.56%), with most being internal microdeletions. Unexpectedly, the microdeletions did not result in loss of their gene products. Screening PDE4D expression in 11 different types of primary tumor samples (n = 165) with immunohistochemistry staining revealed that its protein levels were up-regulated compared with corresponding nontransformed tissues. Importantly, depletion of endogenous PDE4D with three independent shRNAs caused apoptosis and growth inhibition in multiple types of cancer cells, including breast, lung, ovary, endometrium, gastric, and melanoma, which could be rescued by reexpression of PDE4D. We further showed that antitumor events triggered by PDE4D suppression were lineage-dependently associated with Bcl-2 interacting mediator of cell death (BIM) induction and microphthalmia-associated transcription factor (MITF) downregulation. Furthermore, ectopic expression of the PDE4D short isoform, PDE4D2, enhanced the proliferation of cancer cells both in vitro and in vivo. Moreover, treatment of cancer cells with a unique specific PDE4D inhibitor, 26B, triggered massive cell death and growth retardation. Notably, these antineoplastic effects induced by either shRNAs or small molecule occurred preferentially in cancer cells but not in nonmalignant epithelial cells. These results suggest that although targeted by genomic homozygous microdeletions, PDE4D functions as a tumor-promoting factor and represents a unique targetable enzyme of cancer cells.

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Recombinant Expression of a Type IV, cAMP-Specific Phosphodiesterase: Characterization and Structure−Function Studies of Deletion Mutants

Cited by 52 publications

References 47 publications

Regulation of T-Cell Activation by Phosphodiesterase 4B2 Requires Its Dynamic Redistribution during Immunological Synapse Formation

Regulation of T-Cell Activation by Phosphodiesterase 4B2 Requires Its Dynamic Redistribution during Immunological Synapse Formation

Expression of an Active, Monomeric Catalytic Domain of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)

Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers

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