The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain Marco Gross-Langenhoff 1 , Arnulf Stenzl 2 , Florian Altenberend 1 , Anita Schultz 1 and Joachim E. Schultz 1 1 Pharmazeutisches Institut, Universita ¨ tTu ¨ bingen, Germany 2 Urologische Universita ¨ tsklinik, Universita ¨ tTu ¨ bingen, Germany The secondary messengers cAMP and cGMP regulate a variety of signalling pathways in essentially all cells [1]. Their intracellular levels are balanced by the rates of biosynthesis via adenylyl cyclase (EC 4.6.1.1) and guanylyl cyclase (EC 4.6.1.2) and of breakdown via cyclic nucleotide phosphodiesterase (PDEs; EC 3.1.4.17). The human genome codes for 10 ade- nylyl cyclase (AC) and 21 PDE genes. The latter have been grouped into 11 families based on sequence simi- larities [2]. This set of biosynthetic and degrading enzymes allows precise regulation of secondary mes- senger levels in different tissues, in individual cell types and in different subcellular compartments. As far as PDEs are concerned, all share a highly conserved cata- lytic domain located C-terminally, yet show consider- able variability in their N-terminal regions where the regulatory domains reside [3,4]. PDE1, )2, )4, )5, )6, )10 and )11 have an N-terminal tandem domain arrangement, and this may exert its regulatory control of the catalytic domains in a mechanistically similar mode. PDE2, )5, )6, )10 and )11 have N-terminal tandem GAF domains preceded by N-terminals of different lengths. GAF domains are small-molecule- binding domains that have been identified in > 3000 proteins throughout all taxonomic kingdoms [5,6]. The acronym GAF is derived from the proteins in which these domains were initially identified (mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases and Keywords adenyl cyclase; cGMP; cyclic nucleotide phosphodiesterase 11; GAF tandem domain; regulation Correspondence J. E. Schultz, Pharmazeutisches Institut, Universita ¨ tTu ¨ bingen, Morgenstelle 8, 72076 Tu ¨ bingen, Germany Fax: +49 7071 295952 Tel: +49 7071 2974676 E-mail: joachim.schultz@uni-tuebingen.de (Received 22 October 2007, revised 4 December 2007, accepted 5 February 2008) doi:10.1111/j.1742-4658.2008.06319.x The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 lm cGMP for half-maximal effective concentration (EC 50 )ofa cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we exam- ine whether modifications in the N-terminus of PDE11A4 affect cGMP sig- nalling. The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases (Ser117, Ser168). Phosphoryla- tion of both by cAMP-dependent protein kinase decreased the EC 50 value for cGMP from 72 to 23 lm. Phosphomimetic point mutations (S117D ⁄ S167D), which project complete phosphorylation, lowered the EC 50 value to 16 lm. Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC 50 value of 7.6 lm. Truncation by 176 amino acids was required for cGMP EC 50 values to decrease to below 10 lm; a construct truncated by 168 amino acids had an EC 50 value of 224 lm. The decrease in EC 50 values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however. We conclude that N-ter- minal modifications strongly affect cGMP regulation of hPDE11A4. Abbreviations AC, adenylyl cyclase; cAK, cAMP-dependent protein kinase; cGK, cGMP-dependent protein kinase; EC 50 , half-maximal effective concentration; hPDE11A4, human PDE11A4; PDE, cyclic nucleotide phosphodiesterase. FEBS Journal 275 (2008) 1643–1650 ª 2008 The Authors Journal compilation ª 2008 FEBS 1643 Escherichia coli transcription factor FhlA) [7]. cGMP was found to be a ligand for the GAF domains of PDE2 [8,9], PDE5 [10], PDE6 [11] and PDE11 [12], whereas cAMP regulates PDE10 [12]. Here we deal with PDE11A4, a splice variant of the most recently discovered PDE family [13–16]. The four PDE11 isoforms differ in terms of the length of their N-terminal domains. Only PDE11A4 contains a com- plete N-terminal tandem GAF ensemble and an addi- tional 196 amino acid N-terminus [13]. Within this extended N-terminus in vitro phosphorylation sites have been identified at Ser117 and Ser162 [13]. In vivo phosphorylation and a potential physiological role for these phosphorylation sites have not been reported. PDE11 is a true dual-substrate PDE, i.e. it hydro- lyses cAMP and cGMP with similar K m and V max val- ues [13–15,17]. The tissue distribution of the PDE11 isozymes has not yet been fully examined. It is known that robust expression in humans occurs in the pros- tate [18,19], testis [20–22], spermatozoa [22] and brain [23]. The physiological role of PDE11 in the regulation of cyclic nucleotide levels is currently unclear, partly because of a lack of any clear pattern in its tissue dis- tribution and partly because a PDE11-specific inhibitor has not yet been developed. In studies using a PDE11 knockout mouse model, it has been discussed in terms of its involvement in sperm development and function [22]. In addition, it is speculated that PDE11 plays a role in the pathology of major depressive disorder [23] and the development of endocrinal tumours [24]. Fur- thermore, hPDE11 presumably plays a role in various urological diseases such as benign prostatic syndrome, erectile dysfunction and premature ejaculation [25,26]. In agreement with the expression of PDE5 and PDE11 in the transitional zone of the prostate, these PDE iso- forms might also have a role in regulating the prolifer- ation of glandular epithelial tissue; PDE5 and PDE11 inhibitors might, therefore, prevent the malignant transformation of prostatic cells. In recent studies, we used a chimera between the hPDE11 GAF tandem domain and cyanobacterial ade- nylyl cyclase cyaB1, and identified cGMP as a ligand for the GAF domain [12]. However, 72.5 lm cGMP is required for half-maximal activation of the AC via the GAF-A domain of the tandem, a concentration out- side the physiological range. This stimulation depends on the presence of the hPDE11A4 N-terminus because a construct in which the N-terminus is replaced by that of the cyaB1 AC, is not stimulated [12]. In another study with the PDE5 tandem GAF ensemble, we dem- onstrated that the N-terminus profoundly affects GAF signalling [27]. We therefore probed whether the 196- residue N-terminus which precedes the hPDE11A4 GAF tandem affects intramolecular signalling. The data indicate that phosphorylation and ⁄ or proteolytic processing of the N-terminus substantially increases cGMP affinity and may be important in hPDE11A4 regulation. Results Effect of phosphorylation at Ser117 and Ser162 Previously, we have shown that modifications, e.g. phosphorylation, within the 148-amino acid N-termi- nus of PDE5 affect intramolecular cGMP signalling toward the cyanobacterial reporter enzyme cyaB1 AC [27]. PDE11A4 has an N-terminus of 196 amino acids, i.e. other than PDE2 (221 N-terminal residues) it has the longest N-terminus of human GAF-containing PDE2, )5, )6, )10 and )11. With 27 strongly basic amino acids, Arg and Lys, it has a calculated isoelec- tric point of 10.8 and carries 10 positive charges at pH 7, i.e. this N-terminus may be prone to interact with other subdomains. For comparison, the N-termini of PDE2 and PDE5 have isoelectric points of 5.3 and 5.7, respectively. The PDE11A4 N-terminus has two phosphorylation sites, Ser117 and Ser162 imbedded in the signature sequences RRA 117 S for cAMP-dependent protein kinase (cAK; RRXS) and RKA 162 S for cGMP-dependent protein kinase (cGK; RKXS). It has previously been shown that both sites are phosphory- lated in vitro by cAK or cGK, yet the functional con- sequences have not been reported [13]. We used the catalytic subunit of cAK for phosphorylation of the chimeric protein and observed that the EC 50 for cGMP stimulation of cyaB1 was reduced to 23 ± 1.2 lm, whereas basal activity (7.4 and 6.8 nmoles cAMPÆ mg )1 Æmg )1 for the unphosphorylated and phosphory- lated proteins, respectively) and the extent of stimula- tion were unaffected (n = 2; data not shown). This reduction was due to phosphorylation within the N-terminus because, as we have shown previously, the reporter enzyme AC is not phosphorylated by cAK [27]. However, incubation at 30 °C for 120 min, which was required for phosphorylation, resulted in a consid- erable loss of AC activity, as apparent in the respective control incubations, and precluded further detailed studies. Therefore, we used phosphomimetic mutations and replaced both residues with either aspartate or glu- tamate. Single mutations of Ser117 (S117D and S117E) or Ser162 (S162D and S1162E) resulted in slight increases in cGMP efficiency which did not reach statistical significance (Table 1). However, in the double-mutant S117D ⁄ S162D, cGMP efficacy was enhanced and the EC 50 value for cGMP decreased Regulation of PDE11 GAF domains M. Gross-Langenhoff et al. 1644 FEBS Journal 275 (2008) 1643–1650 ª 2008 The Authors Journal compilation ª 2008 FEBS significantly to 16 lm (Table 1). The extent of cGMP stimulation was significantly reduced in all but one of the phosphomimetic mutants compared with unphos- phorylated wild-type protein. The reduction in maxi- mal cGMP stimulation of the S117D ⁄ S162D mutant was not due to an increase in basal activity (7 nmoles cAMPÆmg )1 Æmg )1 ). cAMP did not stimulate (data not shown). Similarly, the K m values for ATP and the V max values were unaffected by the mutations. The PDE11A4 N-terminus affects cGMP regulation We have shown that a chimera consisting of the PDE11A4 tandem GAF domain and the cyaB1 N-ter- minus in front of cyab1 AC, is not stimulated by cGMP or cAMP [22]. This indicated that the N-termi- nus which precedes the GAF tandem domain either affects intramolecular signal transduction and ⁄ or directly inhibits the C-terminally located AC. Based on two available GAF tandem structures and correspond- ingly adapted sequence alignments, we designated Lys196 as the last residue of the N-terminus and the start of GAF-A at Lys197, the position at which a short a helix that is visible in both structures starts [24,28]. Accordingly, we generated two shortened chimeras, one starting at Lys197 [PDE11A4(197- 568)cyaB1 AC] and one at Lys198 [PDE11A4(198- 568)cyaB1 AC]. The data corroborated the aforemen- tioned conclusions because PDE11A4(198-568)cyaB1 AC could not be stimulated by cGMP, whereas PDE11A4(197-568)cyaB1 AC was stimulated 3.4-fold by cGMP with an EC 50 value of 7.6 ± 0.9 lm (n = 10), i.e. the EC 50 value was almost 10-fold lower than that for the full-length construct (Fig. 1). K m val- ues and Hill coefficients (no cooperativity) for both chimeras were comparable, indicating that AC reporter activity was unaffected. Expression of the constructs was comparable, as probed by western blotting of the affinity-purified proteins (Fig. 1). We assume that protein folding was impaired in the shortened PDE11A4(198-568)cyaB1 AC chimera. Next, we asked whether the large reduction in cGMP EC 50 required complete removal of the N-ter- minus or whether it could be accomplished by less rad- ical shortening. A set of N-terminal truncations was planned according to secondary structural predictions (DNA Star Protean). Accordingly, shortened chimeras started with Ser43, Gly110 (prior to the first phosphor- ylation site), Lys119 (just past the first phosphoryla- tion site), Leu149, Leu164 (past the second phosphorylation site), Ala169, Glu177 and Pro187. Table 1. EC 50 values for cGMP and maximal cGMP stimulation for phosphomimetic mutants in human PDE11A4 tandem GAF domain constructs. Chimeric construct EC 50 cGMP (l M) Maximal stimulation (fold) PDE11 ⁄ cyaB1 (wild-type) 72.5 ± 10.1 3.8 ± 0.4 S117D 115.5 ± 26.1 2.3 ± 0.1* S162D 62.9 ± 14.6 2.5 ± 0.5* S117D ⁄ S162D 16.3 ± 5.6* 1.8 ± 0.1* S117E 74.2 ± 2.3 3.6 ± 0.4 S162E 36.4 ± 3.2 2.4 ± 0.2* S117E ⁄ S162E 38.1 ± 1.2 2.6 ± 0.1* *P < 0.05 compared with the parent chimera using Dunnett’s ana- lysis (see text); n = 4–8. 0 40 120 Lys197 –log [cGMP] [M] 6543 35 45 66 116 kDa 80 160 nmol cAMP·mg –1 ·min –1 Lys198 Fig. 1. cGMP dose–response curves of N-terminal shortened PDE11 ⁄ cyaB chimeras for cGMP. Results are means ± SEM (n ‡ 4). s, chimera starting at Lys197 [PDE11A4(197-568)cyaB1 AC]; d, chimera starting with Lys198 [PDE11A4(198- 568)cyaB1 AC]. In several points the error is within the limits of the symbol. (Right) Western blots (0.5 lgÆlane )1 ) of purified proteins. M. Gross-Langenhoff et al. Regulation of PDE11 GAF domains FEBS Journal 275 (2008) 1643–1650 ª 2008 The Authors Journal compilation ª 2008 FEBS 1645 Removal of 42, 109, 118, 148, 163 and 168 N-terminal residues consistently increased basal AC activity (Fig. 2A) and the cGMP concentration needed for half-maximal activation of the attached cyaB1 AC remained high or could not be determined precisely because of low affinity, e.g. when 42 or 109 N-terminal residues were removed (Fig. 2B). Basal activity increased further in constructs starting with Glu177 and Pro187, in conjunction with a distinct increase in cGMP affinity. Thus, removal of at least 176 N-termi- nal amino acids had a dual effect; it released AC from apparent inhibition by its N-terminus and increased the cGMP affinity of the GAF tandem domain up to 20-fold (Figs 1 and 2B). Maximal stimulation by cGMP was significantly affected in three of the nine truncated constructs (Fig. 2C), although the reduction appeared to be minor. By and large, we consider this to be within the experimental variability for this type of assay. Matters were clearly different with regard to the EC 50 values for cGMP stimulation, as determined by dose–response curves for all truncated versions. In fact, the data did not require statistical analysis (Fig. 2C). The EC 50 for cGMP stimulation was 3.5 ± 2.2 lm for the construct beginning at Glu177 and 10.2 ± 4.0 lm for the construct beginning at Pro187 (Fig. 2C). Thus, only the two latter constructs were comparable with that without an N-terminal region, which had an EC 50 value of 7.6 lm cGMP (Fig. 1). cAMP did not stimulate the reporter enzyme significantly in any of these constructs (data not shown). cGMP-binding assays were not carried out because the lm affinities precluded promising experi- ments using this technique. For all shortened con- structs, the K m values for substrate ATP were found to be unchanged and all Hill coefficients were below unity (no cooperativity; data not shown). Because the extent of protein purity after affinity chromatography as eval- uated by SDS ⁄ PAGE and western blots was similar, the data were comparable (Fig. 3D). The slight SDS ⁄ PAGE running inconsistencies (e.g. S43, G110) 0 10 30 50 70 Lys-197 Pro-187 Glu-177 Ala-169 Leu-164 Leu-149 Met-1 Ser-43 Gly-110 Lys-119 0 50 100 150 200 EC 50 -values for cGMP [µM] Met-1 Ser-43 Gly-110 Lys-119 Leu-149 Leu-164 Ala-169 Glu-177 Pro-187 Lys-197 ND ND ND 0 1 2 3 4 Met-1 Ser-43 Gly-110 Lys-119 Leu-149 Leu-164 Lys-197 Ala-169 * Glu-177 * Pro-187 * Maximal stimulation (x-fold) K-198 116 66 45 35 M-1 E-177 A-169 S-43 L-149 K-197 G-11 0 K-11 9 L-164 P-187 nmol cAMP·mg –1 ·min –1 AB C D Fig. 2. The length of the N-terminus of the hPDE11A4 GAF tandem domain affects basal, maximal and cGMP-stimulated cyaB1 AC activity. (A) Basal activities, (B) maximal activities with 3 m M cGMP, and (C) EC 50 values of N-terminally truncated PDE11 ⁄ cyaB AC chimeras. The respective starting amino acids are given on the x-axis. Values are means ± SEM (n ‡ 4). Asterisks in (B) denote significant differences (P < 0.5) from the full-length chimeras starting at Met1. (D) Western blots of purified recombinant proteins used in (A,B). Detection of proteins was with antibody directed against RGS-His4 against the N-terminal affinity tag. Different gels were equalized at the 116-kDa level. Molecular mass markers of the individual SDS ⁄ PAGE gels are on the left for each gel, respectively. Loading of lanes was uniformly with 0.5 lg. Regulation of PDE11 GAF domains M. Gross-Langenhoff et al. 1646 FEBS Journal 275 (2008) 1643–1650 ª 2008 The Authors Journal compilation ª 2008 FEBS were not due to proteolysis because the western blot detected the N-terminal affinity tag and C-terminal degradation at the catalytic domain would have resulted in a loss of AC activity. We noticed that basal AC activities correlated reasonably well with maximal activation which was attainable with cGMP. There- fore, we concluded that intramolecular signalling most likely was unimpaired in the shortened constructs. Discussion Previously we reported that the tandem GAF domains of mammalian PDE2, )5, )10 and )1 functionally cou- ple to the cyanobacterial AC cyaB1 with retention of their regulatory potency [12,27,29]. Thus, cyaB1 AC is regulated by cAMP when coupled to the tandem GAF domain of PDE10, and by cGMP when linked to the tandem GAF domain of PDE2, )5or)11. The bio- chemical properties of signalling by human tandem GAF domains to cyaB1 AC were by and large in agree- ment with data obtained in studies using bacterially expressed GAF domains, e.g. the cyclic nucleotide- binding affinities of PDE2 and )5 GAF domains. How- ever, we were able to determine additional GAF domain properties because of the dissociation of the allosteric regulators, cyclic nucleotides and the substrate of the reporter enzyme, ATP. As far as AC regulation by the PDE11A4 tandem GAF domain is concerned, we have previously reported that cGMP activates with an EC 50 of 72.5 lm [12]. Such a cGMP concentration is reached under only exceptional circumstances, if at all. This may be discussed in two ways: either cGMP regu- lation of PDE11A4 via its N-terminal tandem GAF domain is physiologically irrelevant or potential second- ary modifications of the PDE11A4 tandem GAF will result in increased cGMP sensitivity. Here we examined the second possibility. Effect of phosphorylations at Ser117 and Ser162 In vitro phosphorylation of Ser117 and Ser162 in the hPDE11A4 tandem GAF domain has been reported. A physiological effect on enzyme activity has not yet been reported [13]. Here, serine phosphorylation by the catalytic subunit of cAK, albeit not known whether at position 117, 162 or both, increased cGMP affinity about threefold. We are not aware of the stoi- chiometry of phosphorylation. Because cyaB1 AC activity decreased considerably during incubation with cAK at 37 °C we used phosphomimetic point muta- tions at positions 117 and 162 to evaluate the effect of phosphorylation (Table 1). Single-point mutations at Ser117 to Asp or Glu had no effect, whereas similar ones at Ser162 consistently led to a slight enhancement in cGMP efficacy. It cannot be excluded that individ- ual phosphorylations at these positions might affect additional properties such as intracellular location and translocations, or changes in the interaction profile with other proteins due to charge neutralization of the strongly basic N-terminus. However, phosphomimetic mutations at both positions strongly affected cGMP affinity. With both positions mutated to Asp, the EC 50 value for cGMP was 16.3 lm compared with 72.5 lm in the non-mutated chimera. Therefore, as in PDE5, phosphorylation at the N-terminus of the PDE11A4 GAF tandem may constitute a mechanism regulating the cGMP affinity of the PDE11A4 tandem GAF domain and thus allosterically affect PDE11 activity. The role of the N-terminus of the PDE11A4 GAF tandem domain The N-termini that precede the GAF domains in mammalian PDEs are of significant length, 221 amino acids in PDE2, 148 in PDE5, 82 in PDE10 and 196 in PDE11A4, and it is conceivable that they have a func- tion in conjunction with the regulation of PDE activ- ity in addition to the phosphorylations in PDE5 and PDE11A4 (see above) [27]. Indeed, we have shown that shortening of the PDE5 N-terminus significantly affected intramolecular signalling in chimeras similar to those used in this study. To date, cNMP binding assays using PDE11A4 have been negative and regu- lation by cyclic nucleotides is uncertain [13,15,17], possibly because of the low cGMP affinity of the PDE11A4 GAF domains [12]. Here, we explored whether the PDE11A4 N-terminus is involved in mod- ulating cGMP affinity. We generated a panel of nine N-terminally shortened PDE11A4 tandem GAF chi- meras according to secondary structure predictions. This included the complete removal of 196 amino acids. Interestingly, removal of Lys197 which con- stitutes the start of the first a helix of GAF-A completely abrogated intramolecular signalling, func- tionally validating the sequence alignment. Removal of up to 169 amino acids yielded variable results that might be summarized as a modest increase in basal AC activity and a slight increase in EC 50 values for cGMP. However, removal of 176 amino acids resulted in a profound change, basal AC activity was increased  10-fold and cGMP efficacy was increased  20-fold to an EC 50 value of 3.2 lm, fivefold lower than that observed upon phosphorylation of Ser117 and Ser162 (see above). The kinetic parameters of the attached AC were not altered by N-terminal shortening. One may question whether the biochemical data M. Gross-Langenhoff et al. Regulation of PDE11 GAF domains FEBS Journal 275 (2008) 1643–1650 ª 2008 The Authors Journal compilation ª 2008 FEBS 1647 obtained with the PDE11A4 tandem GA ⁄ cyaB1 AC chimera yields solid information on PDE11A4 holo- enzyme regulation, particularly as no data concerning PDE11A4 regulation are available for direct compari- sons. However, similar chimeras consisting of the cyaB1 AC and the tandem GAF domains of PDE2 or )5 have demonstrated the validity of the approach used here as far as EC 50 values and other parameters involved in PDE regulation are concerned [27,29]. Therefore, one may postulate that N-terminal modifi- cations of the PDE11A4 tandem GAF domains are required to enable cGMP regulation of PDE11 cata- lytic activity. Actually, the terminal region may have two separate effects: one that directly affects cGMP affinity via the GAF domains, and a second compo- nent that acts directly on the catalytic activity. To date, no structure is available for the N-termini of PDE2, )5, )10 or )11 and it will be interesting to see whether common structural features exist in the N-ter- mini of GAF-domain-containing PDEs that might contribute to intramolecular signalling in a similar manner. Another point merits discussion. Irrespective of phosphomimetic mutations or N-terminal shorten- ings, PDE11A4 GAF-tandem-mediated activation of cyaB1 AC was always modest, mostly two- to three- fold, when compared with the effects of the PDE2, PDE5 and PDE10 tandem GAF domains. One may ask whether this is physiologically significant because: (a) a two- to threefold change in the V max value will hasten intracellular adjustment of cAMP or cGMP levels considerably and thus possibly shorten excited cell states; (b) regulation of PDE4 isozymes is reported to involve the phosphorylation of a serine located at the beginning of the tandem of upstream conserved regions, which precedes the catalytic domain. PDE4 activation by this phosphorylation is about twofold, i.e. in the same order of magnitude as that reported here for PDE11A4 [4]; and (c) it was observed that removal of the N-terminal portion in long forms of PDE4 resulted in an increase in basal PDE activity [4], a situation not unlike that observed here and also reported for the PDE5 GAF tandem [27]. Finally, one may ask whether phosphorylation at Ser117 and Ser162 and N-terminal shortening are a sin- gle mechanism or two independent control mechanisms. We generated phosphomimetic mutations in the trun- cated constructs beginning at Ser43 and Lys119. In these chimeras, the EC 50 values for cGMP stimulation were either too high to be determined or > 100 lm (data not shown). Therefore, it seems that we were deal- ing with two independent control mechanisms. In sum- mary, the data emphasize the important role that the N-termini of those PDEs possessing an N-terminal GAF tandem domain may exert in regulation of PDE activity. Obviously, structural information for these extended N-terminal regions would help considerably when discussing the biochemical findings in more detail. Experimental procedures Recombinant DNAs The cyaB1 gene (gi: 15553050) was a gift from M. Ohmori (University of Tokyo, Japan) and a cDNA clone of hPDE11 (gi: 15128482) was provided by G. Quintini (Nycomed, Konstanz, Germany). Throughout, the number- ing of amino acids refers to these genes. The hPDE11A4 1-568 cyaB1 386-859 chimera [12] served as a template to generate all mutants and the N-terminally shortened constructs. Sin- gle- and double-point mutations of PDE11A4 Ser117 and Ser162 were generated by fusion PCR with Pfu DNA poly- merase (Promega, Madison, WI, USA) using corresponding sense and antisense primers (MWG Biotech, Ebersberg, Germany) and nearby restriction sites (KpnI, SacI, StuI and MfeI). N-Terminally shortened constructs were created with respective NdeI sense primers and an MfeI antisense primer in the expression vector pET16b adding a C-terminal His10-tag. To generate PDE 11A4 GAF-A(181-370)cyaB1(386- 859) corresponding parts were amplified by PCR and cloned into pET16b ⁄ pQE30 [27] via BamHI, BglII (GAF- A) and Bgl II, SmaI (cyaB1 AC), respectively. An MRGS- His6-tag was located N-terminally. The hPDE11A4(1-568) construct in pQE60 via 5¢-NcoI and 3¢-BamHI was obtained using hPDE11 as a PCR tem- plate. This added a C-terminal GSRSHis6 affinity tag. The fidelity of all constructs was verified by double stranded sequencing. Primer sequences are available on request. All pQE plasmids were obtained from Qiagen (Hilden, Germany). Expression and purification of recombinant proteins hPDE11A4 ⁄ cyaB1 chimeras were expressed and purified as described earlier [12]. pQE60 constructs were expressed at 16 °C at 400 lm isopropyl b-d-thiogalactoside overnight and the pQE80 constructs at 18 °C and at 1 mm isopropyl b-d-thiogalactoside overnight. Harvested bacteria were stored at )80°C. Adenylyl cyclase assay Activity was assayed for 10 min at 37 °C in 100 lL con- taining 22% glycerol, 50 lg BSA, 50 mm Tris ⁄ HCl pH 7.5, 10 mm MgCl 2 and 75 lm [ 32 P]ATP[aP] (25 kBq; Hart- mann Analytic, Braunschweig, Germany) [30]. We added 2mm [2,8- 3 H]cAMP (150 Bq; GE Healthcare, Freiburg, Regulation of PDE11 GAF domains M. Gross-Langenhoff et al. 1648 FEBS Journal 275 (2008) 1643–1650 ª 2008 The Authors Journal compilation ª 2008 FEBS Germany) was added after stopping the reaction to deter- mine yield during product isolation. The reaction was started by addition of ATP. Substrate conversion was lim- ited to < 10% to ensure linearity. PDE activity was absent in all affinity-purified recombinant proteins. All values are given as mean ± SE. Two-tailed Student’s t-tests were used for statistical evaluation when necessary. Western blot analysis Proteins were mixed with sample buffer and subjected to SDS ⁄ PAGE (12.5%). Proteins were blotted onto poly(vinyli- dene difluoride) membranes and sequentially probed with antibodies directed against either RGS-His4 or His4 (Qiagen) and with a 1 : 5000 dilution of a peroxidase-conjugated goat secondary anti-(mouse IgG) (Dianova, Hamburg, Germany). Peroxidase detection was carried out with the ECL Plus kit (Amersham-Pharmacia, Freiburg, Germany). Preferably, western blots of affinity-purified proteins are depicted because for the current studies it was necessary to ensure that the constructs did not contain degraded products which would affect the cyclase reaction. N-Terminally degraded proteins would not have bound to the Ni 2+ ⁄ nitrilotetra ⁄ acetic acid affinity material. C-Terminally truncated proteins are catalytically inactive, i.e. western blots show the extent of purification of the relevant recombinant protein species. Miscellaneous methods Total protein concentrations were determined using the method described by Bradford [31], with BSA as the stan- dard. Data are given as means ± SEM of between four and eight experiments. 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