Purification and Characterization of the Human PDE4A Catalytic Domain (PDE4A330–723) Expressed in Sf9 Cells

doi:10.1006/abbi.2001.2513

Archives of Biochemistry and Biophysics

Volume 394, Issue 1, 1 October 2001, Pages 54-60

https://doi.org/10.1006/abbi.2001.2513 Get rights and content

Abstract

The human PDE4A catalytic domain (PDE4A^330–723) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration ∼1 mg/ml from light scattering measurement and has a K_m of 2 μM in hydrolyzing cAMP. In comparison, a partially purified PDE4A^330–723 expressed in Escherichia coli has an apparent K_m of 10 μM. The EC₅₀ values for the Mg²⁺- or Co²⁺-mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.

References (27)

M. Conti et al.
Prog. Nucleic Acid Res. Mol. Biol.
(1999)
T.J. Torphy et al.
Pulm. Pharmacol. Ther.
(1999)
J.E. Souness et al.
Immunopharmacology
(2000)
M.D. Houslay et al.
Adv. Pharmacol.
(1998)
C. Sette et al.
J. Biol. Chem.
(1996)
M.B. Beard et al.
J. Biol. Chem.
(2000)
N. Oki et al.
J. Biol. Chem.
(2000)
H. Liu et al.
J. Biol. Chem.
(1999)
S.J. MacKenzie et al.
J. Biol. Chem.
(2000)
G.A. Omburo et al.
Blood
(1997)

M.D. Percival et al.

Biochem. Biophys. Res. Commun.

(1997)

W. Richter et al.

Protein Expr. Purif.

(2000)

M. Salanova et al.

Methods

(1998)

Cited by (8)

Computational investigation of the dynamic control of cAMP signaling by PDE4 isoform types
2022, Biophysical Journal
Cyclic adenosine monophosphate (cAMP) is a generic signaling molecule that, through precise control of its signaling dynamics, exerts distinct cellular effects. Consequently, aberrant cAMP signaling can have detrimental effects. Phosphodiesterase 4 (PDE4) enzymes profoundly control cAMP signaling and comprise different isoform types wherein enzymatic activity is modulated by differential feedback mechanisms. Because these feedback dynamics are non-linear and occur coincidentally, their effects are difficult to examine experimentally but can be well simulated computationally. Through understanding the role of PDE4 isoform types in regulating cAMP signaling, PDE4-targeted therapeutic strategies can be better specified. Here, we established a computational model to study how feedback mechanisms on different PDE4 isoform types lead to dynamic, isoform-specific control of cAMP signaling. Ordinary differential equations describing cAMP dynamics were implemented in the VirtualCell environment. Simulations indicated that long PDE4 isoforms exert the most profound control on oscillatory cAMP signaling, as opposed to the PDE4-mediated control of single cAMP input pulses. Moreover, elevating cAMP levels or decreasing PDE4 levels revealed different effects on downstream signaling. Together these results underline that cAMP signaling is distinctly regulated by different PDE4 isoform types and that this isoform specificity should be considered in both computational and experimental follow-up studies to better define PDE4 enzymes as therapeutic targets in diseases in which cAMP signaling is aberrant.
The measurement of cyclic nucleotide phosphodiesterase 4 activities via the quantification of inorganic phosphate with malachite green
2009, Analytica Chimica Acta
Citation Excerpt :
DMSO and methanol had negligible effects at final contents below 0.5% (Table 3). By Lineweaver–Burk plot analysis, the resultant Km of PDE4 at pH 7.4 was (8.6 ± 0.2) (n = 2) (Fig. 6), which was exactly consistent with that for the recombinant human PDE4 expressed in E. coli[18–20]. cAMP at 60.0 μM was then used to estimate the half-inhibition concentration (IC50) of rolipram on PDE4, which benefited the detection of common types of reversible inhibitors on PDE4 including the rare uncompetitive inhibitors [27,28].
A spectrometric method was investigated to measure the activities of recombinant human cyclic nucleotide phosphodiesterase 4 (PDE4), based on the use of malachite green (MLG) to quantify phosphate released from adenosine-5′-monophosphate (AMP) by the action of calf intestinal alkaline phosphatase (CIAP). Glycerol at 2% stabilized the complex between MLG and phosphomolybdate, whose absorbance at 630 nm was proportional to phosphate concentrations with resistance to common substances in PDE4 reaction mixtures except papaverine. CIAP had the Michaelis–Menten constant (K_m) of (12.0 ± 2.1) μM (n = 3) for AMP at pH 7.4, and was resistant to EDTA below 0.20 mM. By the coupled end-point assay at 30.0 U L⁻¹ CIAP with reaction durations within 30 min, the rates to release phosphate in PDE4 reaction mixtures containing 10.0 mM MgCl₂ and 0.10 mM EDTA linearly responded to the amounts of PDE4 over wide ranges. Meanwhile, K_m of PDE4 was (8.8 ± 0.2) μM (n = 2), zinc ion inhibited PDE4 and rolipram had the inhibition constant about 10 nM. These results supported that by the coupled end-point assay, this method was promising to screen of PDE inhibitors that had no interference with the MLG assay of phosphate.
Three-dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity
2003, Structure
Selective inhibitors against the 11 families of cyclic nucleotide phosphodiesterases (PDEs) are used to treat various human diseases. How the inhibitors selectively bind the conserved PDE catalytic domains is unknown. The crystal structures of the PDE4D2 catalytic domain in complex with (R)- or (R,S)-rolipram suggest that inhibitor selectivity is determined by the chemical nature of amino acids and subtle conformational changes of the binding pockets. The conformational states of Gln369 in PDE4D2 may play a key role in inhibitor recognition. The corresponding Y329S mutation in PDE7 may lead to loss of the hydrogen bonds between rolipram and Gln369 and is thus a possible reason explaining PDE7's insensitivity to rolipram inhibition. Docking of the PDE5 inhibitor sildenafil into the PDE4 catalytic pocket further helps understand inhibitor selectivity.
Dimerization of the type 4 cAMP-specific phosphodiesterases is mediated by the upstream conserved regions (UCRs)
2002, Journal of Biological Chemistry
Citation Excerpt :
Further experiments are, however, required to confirm this possibility as PDE3A/B catalytic domains appear to behave as dimers (39). At odds with the data reported on PDE5, it was thought that PDE4 oligomerization is mediated by several different domains localized within the C terminus or the catalytic domain (40-44). On the basis of deletion mutants expressed in bacteria, a previous report had reached the conclusion that the C terminus mediates dimerization of a short PDE4D splice form (41).
The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.
In vitro PKA phosphorylation-mediated human PDE4A4 activation
2002, FEBS Letters
The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg²⁺ dose–response (EC₅₀ of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme’s sensitivity to Mg²⁺, leading to 4-fold increased cAMP hydrolysis without affecting its K_m. The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.
The molecular biology of phosphodiesterase 4 enzymes as pharmacological targets: An interplay of isoforms, conformational states, and inhibitors<sup>s</sup>
2021, Pharmacological Reviews

View all citing articles on Scopus

¹: To whom correspondence and reprint requests should be addressed. Fax: (514) 428-4900. E-mail: [email protected].

View full text

Regular ArticlePurification and Characterization of the Human PDE4A Catalytic Domain (PDE4A330–723) Expressed in Sf9 Cells

Abstract

Prog. Nucleic Acid Res. Mol. Biol.

Pulm. Pharmacol. Ther.

Immunopharmacology

Adv. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Blood

Biochem. Biophys. Res. Commun.

Protein Expr. Purif.

Methods

Regular Article
Purification and Characterization of the Human PDE4A Catalytic Domain (PDE4A^330–723) Expressed in Sf9 Cells