Sub-family selective actions in the ability of Erk2 MAP kinase to phosphorylate and regulate the activity of PDE4 cyclic AMP-specific phosphodiesterases

Abstract

Expressed in intact cells and in vitro, PDE4B and PDE4C isoenzymes of cyclic nucleotide phosphodiesterase (PDE), in common with PDE4D isoenzymes, are shown to provide substrates for C-terminal catalytic unit phosphorylation by the extracellular signal-regulated kinase Erk2 (p42(MAPK)). In contrast, PDE4A isoenzymes do not provide substrates for C-terminal catalytic unit phosphorylation by Erk2. Mutant PDE4 enzymes were generated to show that Erk2 phosphorylation occurs at a single, cognate serine residue located within the C-terminal portion of the PDE4 catalytic unit. PDE4 long-form isoenzymes were markedly inhibited by Erk2 phosphorylation. The short-form PDE4B2 isoenzyme was activated by Erk2 phosphorylation. These functional changes in PDE activity were mimicked by mutation of the target serine for Erk2 phosphorylation to the negatively charged amino acid, aspartic acid. Epidermal growth factor (EGF) challenge caused diametrically opposed changes in cyclic AMP levels in COS1 cells transfected to express the long PDE4B1 isoenzyme compared to cells expressing the short PDE4B2 isoenzyme. We suggest that PDE4 enzymes may provide a pivotal point for integrating cyclic AMP and Erk signal transduction in cells with 4 genes encoding enzymes that are either insensitive to Erk2 action or may either be activated or inhibited. This indicates that PDE4 isoenzymes have distinct functional roles, giving credence to the notion that distinct therapeutic benefits may accrue using either PDE4 subfamily or isoenzyme-selective inhibitors.

Figure 1

Action of EGF on COS cells expressing long PDE4 isoenzymes. COS1 cells were transfected with the indicated long PDE4 isoenzyme and challenged with EGF (50 ng ml⁻¹) in the absence or presence of the Mek inhibitor, PD98059 (20 μM), or the PKA inhibitor, H89 (0.5 μM), for the indicated time prior to harvesting for the determination of PDE4 activity. Data are mean±s.d. for three separate experiments. Various long isoenzymes, namely (a) PDE4B1, (b) PDE4C2 and (c) PDE4A8, were used in these studies, with activities expressed relative to that in the untreated control cells (100%) which were 350–410, 510–620 and 800–870 pmol min⁻¹ mg⁻¹ protein (range; n=3) for PDE4B1, PDE4C2 and PDE4A8, respectively.

Figure 2

Erk2 phosphorylation of PDE4 isoenzymes. As detailed in Methods, the indicated recombinant PDE4 isoenzymes were immunoprecipitated and then incubated with Erk2 under phosphorylating conditions in vitro and then subjected to SDS–PAGE with visualization by phosphorimager. The arrows indicate the expected position for migration of these species. This was confirmed by immunoblotting (not shown). Data are typical of experiments done at least three times. In all cases 150 μg of transfected COS1 cell lysate was taken for the phosphorylation analyses. The various PDE4 species were immunoprecipitated using class-specific antibodies directed at epitopes within their unique C-terminal regions. Tracks are of samples with Erk2 addition to the phosphorylation experiment unless indicated as ‘nk'–no kinase (Erk2) addition. Wild type enzymes were used except that in some instances mutant species were used where the putative serine target for Erk2 action was replaced with alanine. The various isoenzymes and constructs used were: (a) 104-kDa long PDE4B1 (B1) also showing the Ser⁶⁵⁹Ala mutation (ala); (b) 80-kDa long PDE4C2 (C2) with Ser⁵³⁵Ala (ala) mutant; (c) 98-kDa long PDE4A8 (A8); (d) 80-kDa short PDE4B2 (B2) plus the Ser⁴⁸⁷Ala mutant (ala) and (e) 79-kDa short PDE4A1 (A1). These data are typical of experiments done at least three times.

Figure 3

Action of EGF on the activity of short PDE4B2 and PDE4A1 isoenzymes. Experiments were done as per the legend for Figure 2. Shown here are the (a) PDE4B2, (b) PDE4A1 short isoenzymes. Activities are shown relative to those of the untreated controls (100%) with mean±s.d. (n=3 separate experiments shown). The specific activities of the lysates for COS cells expressing these species were 840–950 and 1150–1220 pmol min⁻¹ mg⁻¹ protein, respectively, for the species listed above. In addition, we show (c) the effect of EGF treatment on the endogenously expressed short PDE4B2 form found in U937 monocytic cells in the absence and presence of PD98059 (20 μM), the inhibitor of Mek activation. These data are typical of experiments done at least three times.

Figure 4

EGF-mediated changes in COS cell cyclic AMP levels. COS1 cells were transfected to express either the long PDE4B1 isoenzyme or the short PDE4B2 isoenzyme. They were then challenged with EGF (50 ng ml⁻¹) and harvested at the indicated time for determination of intracellular cyclic AMP concentration. Data are shown for cells that had been incubated in the absence or presence of PD98059 (20 μM). In untransfected or empty vector-transfected cells EGF did not elicit any change in COS cell cyclic AMP levels. Data represent mean±s.d. of n=3 separate experiments. In the absence of EGF the intracellular cyclic AMP concentrations were 0.91 pmol 10⁻⁶ cells in PDE4B1-expressing cells and 1.2 pmol 10⁻⁶ cells in PDE4B2-expressing cells. These data are typical of experiments done at least three times.