Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in Upstream Conserved Region 1 (UCR1)

Abstract

1. Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP-specific phosphodiesterase long isoforms. 2. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. 3. The PKA inhibitor, H89, ablated forskolin activation of wild-type long PDE4 isoforms. 4. Activated PKA caused the in vitro phosphorylation of recombinant wild-type long PDE4 isoforms, but not those where the STR was mutated to alanine. 5. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin-challenged cells treated with H89. Neither was it evident in forskolin-challenged cells expressing long isoforms where the STR had been mutated to alanine. 6. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform-specific N-terminal region. 7. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. 8. Long PDE4 isoforms from all four sub-families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed.

Figure 1

Expression of PDE4 isoforms in transfected COS1 cells. Shows immunoblots of COS1 cells transfected to express PDE4A8, PDE4B1, PDE4C2 and PDE4D5 long isoforms. Detection was performed using antisera specific for the particular PDE4 sub-class. In each instance a single immunoreactive species (arrowed), of the appropriate molecular weight, was identified using transfected cells. These were 98 kDa for PDE4A8, 104 kDa for PDE4B1, 80 kDa for PDE4C2 and 105 kDa for PDE4D5. Data are shown here for the indicated transfected cells that either had (+fsk) or had not (C) been treated with forskolin (100 μM) together with the non-selective, low affinity PDE inhibitor, IBMX (100 μM) for 15 min prior to harvesting. The data shown are typical of experiments done at least three times with different transfections.

Figure 2

Activation of PDE4 long isoforms in forskolin-challenged cells. COS1 cells, transfected to express the indicated PDE4 isoforms, were challenged with the adenylyl cyclase activator, forskolin (100 μM), together with the non-specific PDE inhibitor, IBMX (100 μM). They were then harvested at the indicated time for analysis of PDE4 activity. COS1 cells were transfected with PDE4A8 (a), PDE4A10 (b), PDE4B1 (c), PDE4C2 (d) and PDE4D5 (e). In such cells then >98% of the total PDE activity was due to the transfected species whose activities are given in Table 1. Note that the IC₅₀ value for inhibition of the various PDE4 isoforms by IBMX is around 30 μM. Thus, subsequent to cell harvesting, disruption and dilution of extract into the assay, a functionally insignificant level of IBMX (<1 nM) will be carried over into the PDE assay. Where indicated, the PKA selective inhibitor H89 (0.5 μM) was also added with forskolin and IBMX to the cells. As indicated, experiments were also performed using COS1 cells that were transfected to express various mutant PDE4 isoforms where the putative serine target in UCR1 for phosphorylation by PKA had been replaced with an alanine residue. These were Ser⁸⁹Ala-PDE4A8, Ser¹³³Ala-PDE4B1, Ser¹³Ala-PDE4C2 and Ser¹²⁶Ala-PDE4D5. In addition to this the Thr⁸³Ala-PDE4D5 mutant was also evaluated in order to delete a potential PKA phosphorylation site within the unique N-terminal region of PDE4D5. Activities are shown as a percentage relative to that of the control, untreated form. Data are given as means±s.d. for n=3 experiments done using separate transfections.

Figure 3

Phosphorylation of long PDE4 isoforms by PKA. COS1 cells were transfected to express various long PDE4 isoforms. Lysates containing the indicated recombinant wild-type (wt) forms were then treated with a phosphorylation mixture containing radiolabelled ³²P-ATP in either the presence or absence of the activated catalytic unit of PKA. They were then subjected to SDS – PAGE with detection using a phosphorimager. In the presence of PKA then a radiolabelled species of the appropriate size, namely 98 kDa for PDE4A8 (a), 104 kDa for PDE4B1 (b), 80 kDa for PDE4C2 (c) and 105 kDa for PDE4D5 (d), was obtained in each instance. However, no such species were apparent for samples that had not been treated with PKA catalytic unit. Ser⁸⁹Ala-PDE4A8 (a), Ser¹³³Ala-PDE4B1 (b), Ser¹³Ala-PDE4C2 (c) and Ser¹²⁶Ala-PDE4D5 (d) PDE4 isoforms were also used, as indicated, where the putative serine target site in UCR1 for phosphorylation by PKA had been mutated to alanine. In these instances data are shown for experiments where active PKA catalytic unit was added. Also shown (e) is data for the treatment, with activated PKA, of the Thr⁸³Ala-PDE4D5 mutant form where the potential PKA phosphorylation site in the unique N-terminal region of PDE4D5 was mutated to alanine. In each case, equal immunoreactive amounts of the various PDE4 constructs were used in these experiments, as demonstrated by the immunoblots shown in the right hand panels using antisera specific for the indicated PDE4 sub-family analysed. In the immunoblots tracks 1, 2, 3 analyse, respectively, experiments of wt+PKA, wt  –  PKA and Ala mutant+PKA as in the left hand panel. The data shown are typical of experiments done at least three times with enzymes from different transfections.

Figure 4

Detection of phosphorylated long PDE4 isoforms using a novel antiserum specific for the PKA phosphorylated serine residue in UCR1. These data show the results of immunoblots done with the PS54-UCR1-A1 antiserum. COS-1 cells were transfected with the indicated PDE4 isoform and then harvested and analysed by Western blotting. In (a) cells were transfected with PDE4D3 and then either challenged or not, as indicated, with forskolin (100 μM) together with IBMX (100 μM) for 15 min. In one instance the antiserum was treated with a 10-fold excess of the peptide used to generate it in order to block interaction. The single immunoreactive species identified in the second lane migrated with immunoreactive PDE4D3 as identified by stripping and re-probing the blots using a PDE4D-specific antiserum (data not shown). In (b) cells were transfected with the indicated PDE4D3 mutant isoform and then either treated (fsk) or not with forskolin (100 μM) together with IBMX (100 μM), followed by harvesting and Western blotting. The mutants used were serine to alanine replacements as indicated. In (c) cells were transfected to express the indicated PDE4 long isoforms, either as wild-type or with the putative PKA target serine in UCR1 mutated to alanine. Cells were then treated or not with forskolin (100 μM) together with IBMX (100 μM), as indicated, for 15 min prior to harvesting and Western blotting. In some instances cells were additionally treated with H89 (0.5 μM) together with forskolin and IBMX. The single immunoreactive species identified in forskolin-treated cells transfected to express either PDE4A8 or PDE4B1 or PDE4C2 or PDE4D5 migrated as expected for these isoforms (see legend to Figure 1). In each comparative experiment we ensured that equal amounts of the indicated PDE4 long isoform was present. This was gauged by immunoblotting lysates with a PDE4 subfamily-specific antiserum in each case. This was then confirmed when the blots were stripped and re-probed for the appropriate isoform (data not shown). The data shown are representative of at least three separate experiments using different transfections.

Figure 5

The PKA-mediated mobility shift of PDE4D3 requires phosphorylation at both Ser¹³ and Ser⁵⁴. In (a) COS-1 cells were transfected to express the indicated wild-type or mutant forms of PDE4D3. They were then either treated (+fsk) or not (C) with forskolin (100 μM) together with IBMX (100 μM) for 10 min prior to harvesting. They were then identified using a PDE4D specific antiserum. In (b) is shown an experiment where cells were treated with forskolin together with IBMX for 10 min prior to harvesting and then samples were either treated with alkaline phosphatase (+ptase) or not (C) as described in Methods. The data shown are representative of at least three separate experiments using different transfections.

Figure 6

Time dependence of the PKA-induced mobility shift of PDE4D3 determined using novel antisera specific for the PKA phosphorylated serine residue in UCR1 and for Ser¹³ in the unique N-terminal region of PDE4D3. Panel (a) shows a time-course of the effect of challenging COS-1 cells transfected to express PDE4D3 with forskolin (100 μM) together with IBMX (100 μM). At the indicated times cells were harvested and then immunoblotted with antiserum specific for PDE4D (upper lane), with the PS54-UCR1-A1 antiserum (middle lane) and also with the PS13-4D3-A1 antiserum (lower lane). Lanes show data for equal loading of immunoreactive PDE4D3 in all experiments. The data shown are representative of at least three separate experiments using different transfections. Panel (b) shows the use of the novel polyclonal antiserum, PS13-4D3-A1, to detect the PKA-phosphorylated form of Ser¹³ in PDE4D3. Shown are lysates of transfected COS cells immunoblotted using this antiserum. COS1 cells transfected to express wild type PDE4D3 (wt) were either challenged (+fsk) or not (ctr) with forskolin together with IBMX for 10 min prior to harvesting. The single immunoreactive band identified in ‘+fsk' treated cells was identified as PDE4D3 by stripping and re-probing the blots with PDE4D-specific antiserum (data not shown). Also shown are lysates from ‘+fsk' treated PDE4D3 transfected COS1 cells where the polyclonal antiserum, PS13-4D3-A1, had been pre-absorbed with a 10-fold excess of the peptide used to generate it. Panel (c) shows immunoblots, done using antiserum PS13-4D3-A1, of COS1 cells transfected to express the indicated PDE4D3 mutants which were either treated (fsk) or not, as indicated, with forskolin together with IBMX for 10 min prior to harvesting. Used were wild-type PDE4D3 (wt), the Ser¹³Ala mutant, the Ser⁵⁴Ala mutant and the Ser¹³Ala : Ser⁵⁴Ala double mutant of PDE4D3. The data shown are representative of at least three separate experiments using different transfections.

Figure 7

Rolipram inhibition of PKA-phosphorylated long PDE4 isoforms. High speed supernatant/soluble (S2) fractions were made from COS1 cells transfected to express the indicated PDE4 isoforms that either had or had not been treated with forskolin (100 μM) together with IBMX (100 μM) for 10 min prior to harvesting. The inhibitory effect of rolipram on the PDE activity was assessed using 1 μM cAMP as substrate. The indicated enzymes were (a) PDE4A10, (b) PDE4A8, (c) PDE4B1, (d) PDE4C2, (e) PDE4D5 and (f) PDE4D3. Activity of the various PDE4 species in transfected COS cells was as indicated in Table 1 and in Figure 2. The data presented give mean±s.d. of three separate experiments using different transfections.