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Am J Physiol Gastrointest Liver Physiol 294: G327-G335, 2008. First published November 15, 2007; doi:10.1152/ajpgi.00430.2007
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NEUROREGULATION AND MOTILITY

Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A

Karnam S. Murthy and Wimolpak Sriwai

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 21 September 2007 ; accepted in final form 12 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of G{alpha}q minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-β-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.

phosphodiesterases; protein kinase A; muscle relaxation

SMOOTH MUSCLE TONE IS MODULATED by the release of excitatory neurotransmitters and inhibitory neurotransmitters. Excitatory transmitters (e.g., acetylcholine) initiate distinct signaling cascades involving activation of myosin light chain (MLC) kinase, protein kinase C (PKC), and Rho kinase. 20-kDa MLC (MLC20) phosphorylation and contraction are initiated via Ca2+/calmodulin-dependent activation of MLC kinase and maintained by PKC and Rho kinase via inhibition of MLC phosphatase activity (27, 29). Inhibitory transmitters [nitric oxide, vasoactive intestinal peptide (VIP), and its homologue pituitary adenylyl cyclase activating peptide (PACAP)] initiate signaling cascades that ultimately lead to concurrent generation of cAMP and cGMP and activation of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (28). Both PKA and PKG act on various loci in the signaling cascade that mediate contraction resulting in inhibition of cytosolic Ca2+ and MLC20 phosphorylation leading to muscle relaxation (27, 28). Cyclic nucleotide-mediated signaling is terminated by the actions of phosphodiesterases (PDEs), which provide the only means of degrading cAMP and cGMP levels (4, 13). Thus the activation state of PDEs plays a critical role in determining the strength and duration of cAMP and cGMP signaling.

So far, 11 gene families of cAMP- and cGMP-specific PDEs have been identified; they are classified with respect to their regulation and their substrate specificity (4, 13, 23). Type 4 PDE play a major role in regulating cAMP levels as they exclusively hydrolyze cAMP. Four genes (PDE4A, PDE4B, PDE4C, and PDE4D) encode nearly 20 isozymes of PDE4 family of enzymes (4, 13). The enzymes are categorized into "long" and "short" isozymes on the basis of the presence or absence of two highly conserved regulatory domains, known as upstream conserved region 1 (UCR1) and UCR2, which are unique to PDE4 family, located between the NH2-terminal and catalytic domains (13, 14, 24, 25). The long isozymes have both UCR1 and UCR2, whereas the short isozymes have only UCR2. These two regions interact with each other and regulate the catalytic activity. The posttranslational regulation of PDE4 activity in cells is complex and was first shown for the action of PKA. All long isozymes possess a highly conserved consensus sequence for PKA phosphorylation in UCR1 and phosphorylation of serine in this sequence leads to activation of the enzyme (6, 13, 14, 20, 21, 25, 34, 35). The extent of activation is isozyme specific and ranges from 40% increase in PDE4D4 to 350% in PDE4D7 (33). Phosphorylation of PDE4D3 by PKA also causes increased sensitivity to inhibition by rolipram, a PDE4-specific inhibitor. Phosphorylation of PDE4D3 by PKA on a different NH2-terminal site (Ser13) increases the binding of PDE4D3 with the muscle-selective anchoring/scaffolding protein, mAKAP (20, 21, 25). The short isozymes, which lack UCR1, are not activated by PKA. A consensus site (PQSP) for phosphorylation by ERK is present in the catalytic subunit of all PDE4 subfamilies, except for PDE4A (3, 10, 12, 14, 24). The effect of phosphorylation by ERK on PDE4 activity is determined by the presence or absence of UCR1 and UCR2. The long isozymes containing both UCR1 and UCR2 are inhibited by phosphorylation, whereas short isozymes containing only UCR2 are activated. In long isozymes, however, the inhibition elicited by ERK-mediated phosphorylation was masked by PKA-mediated phosphorylation. Recent studies have identified phosphorylation of a different site (Ser239) in the catalytic domain by an unknown kinase downstream of phosphatidylinositol (PI) 3-kinase in PDE4D3 (11). Phosphorylation at Ser239 alone has no effect on the catalytic activity, but converts the effect ERK-mediated phosphorylation at Ser579 from inhibition to activation of PDE4D3 activity.

In smooth muscle of the gut, the majority of the total cAMP-hydrolyzing activity is provided by PDE3A and PDE4D isozymes, suggesting an important role played by these enzymes in cAMP-mediated signaling (32). Phosphodiesterase 4D5 (PDE4D5) is the main cAMP-specific long PDE4 isozyme expressed in vascular and visceral smooth muscle cells (2). In the present study we tested the hypothesis that constituents in the pathways that mediate contraction could cross-regulate cAMP levels by modulating PKA-stimulated PDE4D5 phosphorylation and activity. Muscle cells were concurrently activated by the contractile agonist cholecystokinin (CCK), which results in activation of PKC to determine whether they cross-regulate PDED4D5 activity and cAMP levels stimulated by forskolin. We have identified a novel mechanism for stimulation of PDE4D5 phosphorylation and activity by CCK. The mechanism involves activation of PKC derived from G{alpha}q/PLC-β-dependent pathway and inhibition of PP2A resulting in augmentation of PDE4D5 phosphorylation. The increase in phosphorylation leads to stimulation of PDE4D5 activity with accompanying decrease in cAMP levels. We suggest that augmentation of PDE4D5 phosphorylation and activity may provide a novel mechanism for cross-regulation of cAMP signaling by contractile agonists.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of dispersed gastric smooth muscle cells. Dispersed gastric smooth muscle cells were prepared as described previously (26, 28). Briefly, strips of circular muscle from rabbit stomach were dissected and incubated at 31°C for 30 min in HEPES medium containing 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 0.6 mM MgCl2, 25 mM HEPES, 14 mM glucose, 2.1% Eagle's essential amino acid mixture, 0.1% collagenase, and 0.1% soybean trypsin inhibitor. After the partly digested strips were washed twice with 50 ml of enzyme-free medium, the muscle cells were allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500-µm Nitex (Tetko, Briarcliff Manor, NY) and centrifuged twice at 350 g for 10 min. For some experiments, muscle cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they attained confluence and were then passaged once for use.

Expression of G{alpha}q minigene in cultured gastric smooth muscle cells. Activation of G{alpha}q was blocked by the expression of cDNA encoding the last COOH-terminal 11 amino acids as described previously (16, 37). The cDNA sequences were amplified by PCR and verified by DNA sequencing. Cultured rabbit gastric smooth muscle cells were transfected transiently with minigene plasmid DNA by use of Effectene transfection reagent. Transfection efficiency (~70%) was monitored microscopically by the coexpression of green fluorescent protein via FITC filters.

Assay for PDE4D5 activity. PDE4D5 activity was measured in immunoprecipitates of PDE4D5 as described previously (32). Muscle cells (1-ml aliquots; 3 x 106 cells/ml) were incubated with the adenylyl cyclase activator forskolin for 5 min in the presence of absence of CCK. PDE4D5 immunoprecipitates were washed in a medium containing 50 mM Tris (pH 7.5), 200 mM NaCl, and 5 mM EDTA and then incubated for 15 min at 30°C in a reaction mixture containing 100 mM MES (pH 7.5), 10 mM EDTA, 0.1 M Mg acetate, 0.9 mg/ml bovine serum albumin, 20 µM cAMP, and [3H]cAMP. The samples were boiled for 3 min, chilled for 3 min, and then incubated at 30°C for 10 min in 20 mM Tris (pH 7.5) medium containing 10 µl of Crotalus atrox snake venom (10 µg/µl). The samples were added to DEAE-Sephacel A-25 columns, and the radioactivity in the effluent was counted. The results were expressed as counts per minute per milligram protein (cpm/mg protein).

In experiments using phosphatases 1 and 2A, the immunoprecipitates were washed with a medium containing 50 mM Tris·HCl (pH 7.5), 0.5 mM EDTA, 5 mM β-mercaptoethanol, and 0.1% Triton X-100 and incubated for 20 min at 30°C with the purified protein phosphatase 1 (0.3 µg) and 2A (0.3 µg). The phosphatases were then removed by further washes with Tris·HCl medium and PDE4D5 phosphorylation and activity was measured.

Phosphorylation of PDE4D5. Phosphorylation of PDE4D5 was measured by immunoblot analysis using phospho-specific antibody (Ser126) as described previously (26, 32). One-milliliter aliquots (3 x 106 cells/ml) of samples were incubated with forskolin and/or CCK for 5 min, the reaction was terminated with an equal volume of lysis buffer, and the samples were placed on ice for 30 min. The cell lysates were separated from the insoluble material by centrifugation at 13,000 g for 15 min at 4°C, precleared with 40 µl of protein A-Sepharose, and incubated with polyclonal PDE4D5 for 2 h at 4°C and with 40 µl of protein A-Sepharose for another 1 h. The immunoprecipitates were washed five times with 1 ml of wash buffer (0.5% Triton X-100, 150 mM NaCl, 10 mM Tris·HCl, pH 7.4), extracted with Laemmli sample buffer, and boiled for 15 min and then separated on 10% SDS-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes. The membranes were incubated for 12 h with phospho-specific antibodies to PDE4D5 (Ser126) and then for 1 h with a horseradish peroxidase-conjugated secondary antibody. The bands were identified by enhanced chemiluminescence (ECL).

PDE4D5 immunoprecipitation and PP1 or PP2A immunoblotting. PDE4D5 immunoprecipitates derived from cells treated with forskolin and/or CCK were separated 10% SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to PVDF membranes as described above. The membranes were incubated for 12 h with antibody to the catalytic subunit of PP1 or PP2A and then for 1 h with a horseradish peroxidase-conjugated secondary antibody. The bands were identified by ECL.

Radioimmunoassay for cAMP by radioimmunoassay. Cyclic AMP production was measured by radioimmunoassay as described previously (28). Briefly, muscle cells (3 x 106 cells) were treated with forskolin in the presence or absence of CCK for 5 min and the reaction terminated with 10% trichloroacetic acid. After extraction with water-saturated diethyl ether, the lyophilized aqueous phase was reconstituted in 500 µl of 50 mM Na acetate (pH 6.2). The samples were acetylated with triethylamine-acetic anhydride (2:1) for 30 min and cAMP was measured in duplicate with 100-µl aliquots. The results were expressed as picomoles per milligram protein.

Materials. [125I]AMP and [3H]cAMP were obtained from Amersham Pharmacia Biotech (Piscataway, NJ); collagenase and soybean trypsin inhibitor were from Worthington Biochemical (Freehold, NJ); Western blot, chromatography material, and protein assay kit were from Bio-Rad Laboratories (Hercules, CA); antibody to PDE4D5 and phospho-antibody to PDE4D5 (Ser126) were obtained from FabGennix (Frisco, TX); protein phosphatase 1 and PP2A catalytic subunit were from Calbiochem (La Jolla, CA); cAMP, Crotalus atrox snake venom, and all other chemicals were from Sigma Chemical (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Augmentation of PDE4D5 phosphorylation and activity by CCK. Treatment of dispersed smooth muscle cells with forskolin (10 µM) induced phosphorylation of PDE4D5 measured using PKA-site (Ser126) phospho-specific antibody (Fig. 1). Previous studies have shown that phosphorylation was blocked by the PKA inhibitor suggesting phosphorylation was mediated via a feedback mechanism (32). Cotreatment of muscle cells with the contractile agonist CCK (1 nM) significantly (P < 0.01) augmented the effect of forskolin on PDE4D5 phosphorylation (Fig. 1).


Figure 1
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  		Fig. 1. Augmentation of forskolin-induced PDE4D5 phosphorylation and activity (A) and attenuation of cAMP levels (B) by CCK. Gastric smooth muscle cells were incubated with forskolin (FSK, 10 µM) or CCK (1 nM) alone or both. Immunoprecipitates derived from 500 µg of protein by use of PDE4D5 antibody were separated on SDS-PAGE and analyzed with phospho-Ser126-specific antibody in the Western blot. Immunoblot analysis with PDE4D5 antibody showed equal amounts of loaded protein. PDE4D5 activity was measured as described in MATERIALS AND METHODS with [3H]cAMP as the substrate. cAMP levels were measured by radioimmunoassay. Values are means ± SE of 4 experiments. **P < 0.01 significant increase in PDE4D5 activity in the presence of CCK compared with activity by forskolin alone. ##P < 0.01 significant decrease in forskolin-induced cAMP levels by CCK.

 
Treatment of dispersed smooth muscle cells with forskolin (10 µM) increased cAMP levels and induced significant activation of PDE4D5 (Fig. 1). Previous studies have shown that the effect of forskolin on PDE4D5 activity was blocked by myristolylated PKI, suggesting forskolin-induced increase in PDED45 activity was mediated by cAMP-activated PKA derived from stimulation of adenylyl cyclase (32). Cotreatment of muscle cells with CCK significantly (P < 0.01) augmented the PDE4D5 activity and attenuated the effect of forskolin on cAMP levels (Fig. 1).

PKC-mediated augmentation of PDE4D5 phosphorylation and activity and inhibition of cAMP levels. The stimulatory effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity was blocked by the phospholipase C (PLC) inhibitor U-73122 (10 µM) and PKC inhibitors GF-109203X (1 µM) or bisindolylmaleimide (1 µM), but not by the RhoA inhibitor C3 exoenzyme (Fig. 2). The MEK inhibitor PD-98059 or the Rho kinase inhibitor Y-27632 had no effect on PDE4D5 phosphorylation induced by forskolin (data not shown). CCK alone in the absence of forskolin also had no effect on PDE4D5 phosphorylation. Previous studies in gastrointestinal smooth muscle have shown that treatment of cells with CCK leads to biphasic increase in PKC activity (31). The initial transient phase reflected diacylglycerol (DAG)-derived from stimulation of G{alpha}q-dependent PLC-β activity, whereas the sustained phase reflected DAG-derived from stimulation of G{alpha}13/RhoA-dependent phospholipase D (PLD) activity. Hydrolysis of phosphatidylcholine by PLD yields phosphatidic acid, which, in turn, is dephosphorylated to yield DAG (31). These results imply that cotreatment with the contractile agonist CCK augmented forskolin-induced PDE4D5 phosphorylation via PKC derived from G{alpha}q-dependent activation of PLC-β1 and that the stimulatory effect of PKC was not due to direct phosphorylation of PDE4D5.


Figure 2
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  		Fig. 2. Augmentation of forskolin-induced PDE4D5 phosphorylation and activity (A) and attenuation of cAMP levels (B) by CCK via PKC derived from PLC-β activation. Gastric smooth muscle cells were incubated with forskolin (10 µM) or forskolin plus CCK (1 nM) in the presence or absence of PLC inhibitor U-73122 (10 µM), PKC inhibitors GF-109203X (1 µM) and bisindolylmaleimide (1 µM), or RhoA inhibitor C3 exoenzyme (2 µg/ml). Immunoprecipitates derived from 500 µg of protein by use of PDE4D5 antibody were separated on SDS-PAGE and analyzed with phospho-Ser126-specific antibody in the Western blot. Immunoblot analysis with PDED5 antibody showed equal amounts of loaded protein. PDE4D5 activity was measured as described in MATERIALS AND METHODS with [3H]cAMP as the substrate. cAMP levels were measured by radioimmunoassay. Values are means ± SE of 4 experiments. **P < 0.01 significant increase in PDE4D5 activity in the presence of CCK compared with activity by forskolin alone. ##P < 0.01 significant decrease in forskolin-induced cAMP levels by CCK.

 
Similarly, the effects of CCK on forskolin-stimulated PDE4D5 activity and cAMP levels were blocked by U-73122 (10 µM), GF-109203X (1 µM), or bisindolylmaleimide (1 µM), whereas C3 exoenzyme had no effect on PDE4D5 activity and cAMP levels. CCK alone in the absence of forskolin had no effect of PDE4D5 activity or cAMP levels. These results imply that cotreatment of muscle cells with CCK augmented cAMP degradation by stimulating PDE4D5 activity via PKC derived from activation of PLC-β.

Treatment of cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 µM) augmented forskolin-induced PDE4D5 phosphorylation and activity and attenuated cAMP levels (Fig. 3).


Figure 3
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  		Fig. 3. Augmentation of forskolin-induced PDE4D5 phosphorylation and activity (A) and attenuation of cAMP levels (B) by PMA. Gastric smooth muscle cells were incubated with forskolin (10 µM) or PMA (1 µM) alone or both. Immunoprecipitates derived from 500 µg of protein by using PDE4D5 antibody were separated on SDS-PAGE and analyzed with phospho-Ser126-specific antibody in the Western blot. Immunoblot analysis with PDE4D5 antibody showed equal amounts of loaded protein. PDE4D5 activity was measured as described in MATERIALS AND METHODS with [3H]cAMP as the substrate. cAMP levels were measured by radioimmunoassay. Values are means ± SE of 4 experiments. **P < 0.01 significant increase in PDE4D5 activity in the presence of PMA compared with activity by forskolin alone. ##P < 0.01 significant decrease in forskolin-induced cAMP levels by PMA.

 
We also confirmed the source of PKC for stimulation of PDE4D5 phosphorylation and activity in cultured muscle cells by expression of G{alpha}q minigene. Cotreatment of cultured muscle cells with CCK augmented forskolin-induced PDE4D5 phosphorylation and PDE4D5 activity and attenuated the increase in cAMP levels. The extent of augmentation of PDE4D5 phosphorylation and activity and attenuation of cAMP levels induced by CCK in cultured muscle cells was similar to that observed in freshly dispersed muscle cells. In cells expressing G{alpha}q minigene, forskolin induced PDE4D5 phosphorylation and increased PDE4D5 activity and AMP levels, and the effect of forskolin was similar to control cells. However, cotreatment of cells with CCK in cells expressing G{alpha}q minigene had no effect on forskolin-induced PDE4D5 phosphorylation and PDE4D5 activity and on forskolin-stimulated cAMP levels (Fig. 4). The results imply that augmentation of forskolin-induced PDE4D5 phosphorylation and activity and inhibition of cAMP levels by CCK are mediated via PKC derived from stimulation of G{alpha}q-dependent PLC-β activity.


Figure 4
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  		Fig. 4. Augmentation of forskolin-induced PDE4D5 phosphorylation and activity (A) and attenuation of cAMP levels (B) by CCK via PKC derived from G{alpha}q-dependent PLC-β activation. Cultured muscle cells expressing vector alone (control) or G{alpha}q minigene were incubated with forskolin (10 µM) or CCK (1 nM) alone or both. Immunoprecipitates derived from 500 µg of protein by use of PDE4D5 antibody were separated on SDS-PAGE and analyzed with phospho-Ser126-specific antibody in the Western blot. Immunoblot analysis with PDE4D5 antibody showed equal amounts of loaded protein. PDE4D5 activity was measured as described in MATERIALS AND METHODS with [3H]cAMP as the substrate. cAMP levels were measured by radioimmunoassay. Values are means ± SE of 4 experiments. **P < 0.01 significant increase in PDE4D5 activity in the presence of CCK compared with activity by forskolin alone. ##P < 0.01 significant decrease in forskolin-induced cAMP levels by CCK.

 
Inhibition of PP2A augments forskolin-induced PDE4D5 phosphorylation and activity. In the absence of direct phosphorylation and activation of PDE4D5 by PKC, we speculated that augmentation of PDE4D5 phosphorylation and activity could be mediated by inhibition of protein phosphatase activity that mediates dephosphorylation of PDE4D5. To examine this notion we used two approaches: in the first approach we examined the selective inhibitor of PP1 or PP2A on forskolin-induced PDE4D5 phosphorylation and activity, and in the second approach we used purified PP1 or PP2A to dephosphorylate PDE4D5 phosphorylation induced by forskolin.

Inhibition of PP2A, but not PP1, mimicked the effect of CCK on PDE4D5 phosphorylation and activity on cAMP levels. PDE4D5 phosphorylation and activity stimulated by forskolin in freshly dispersed muscle cells were significantly augmented by pretreatment of the cells with a low (1 nM) concentration of okadaic acid that selectively inhibits PP2A and by a high concentration of okadaic acid (1 µM) that inhibits both PP1 and PP2A. A low (10 nM) concentration of tautomycin that selectively inhibits PP1 had no effect (Fig. 5).


Figure 5
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  		Fig. 5. Augmentation of forskolin-induced PDE4D5 phosphorylation and activity (A) and attenuation of cAMP levels (B) by PP2A. Gastric smooth muscle cells were incubated with forskolin in the presence or absence of CCK (1 nM), okadaic acid (OA, 1 nM and 1 µM), or tautomycin (Ttm, 10 nM). PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were measured as described in MATERIALS AND METHODS. PDE4D5 activity was expressed as cpm/mg protein. cAMP levels were expressed as pmol/mg protein. Values are means ± SE of 4 experiments. **P < 0.01 significant increase in PDE4D5 activity in the presence of CCK or okadaic acid compared with activity by forskolin alone. ##P < 0.01 significant decrease in forskolin-induced cAMP levels by CCK or okadaic acid.

 
The increase in cAMP levels induced by forskolin in dispersed muscle cells was significantly attenuated by pretreatment of cells with okadaic acid, but not by a low concentration of tautomycin. The results imply that PDE4D5 phosphorylation is selectively dephosphorylated by PP2A and inhibition of PP2A results in the augmentation of PDE4D5 phosphorylation and activity and reduction in the levels of cAMP induced by forskolin.

This is further corroborated by incubating the PDE4D5 immunoprecipitates derived from forskolin-treated cells by with the catalytic subunit of PP1 or PP2A. Forskolin-induced PDE4D5 phosphorylation was dephosphorylated by the catalytic subunit of PP2A, but not PP1 (Fig. 6). We conclude from these studies that PDE4D5 phosphorylation levels are regulated by PP2A activity and inhibition of PP2A activity could lead to augmentation of PDE4D5 phosphorylation.


Figure 6
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  		Fig. 6. Selective dephosphorylation PDE4D5 and inhibition of activity by PP2A catalytic subunit. PDE4D45 immunoprecipitates derived from cells treated with forskolin were incubated with the catalytic subunits of PP1 or PP2A. PDE4D5 phosphorylation and activity was measured as described in MATERIALS AND METHODS. Values are means ± SE of 4 experiments. ##P < 0.01 significant decrease in forskolin-induced PDE4D5 activity in the presence of PP2A catalytic subunit.

 
PKC-mediated decrease in the association of PP2A with phosphorylated PDE4D5. To examine whether PP2A interact with phosphorylated PDE4D5, muscle cells were treated with forskolin in the presence or absence of CCK, and cell lysates were immunoprecipitated by PDE4D5 antibody and then subjected to immunoblot analysis using antibody PP2A catalytic subunit. Treatment of cells with forskolin increased PP2A catalytic subunit in PDE4D5 immunoprecipitates, suggesting association of PP2A with the phosphorylated PDE4D5. In the absence of forskolin (i.e., in the absence of PDE4D5 phosphorylation) there was no association of PP2A with PDE4D5, suggesting that association is subsequent to PDE4D5 phosphorylation by PKA. Cotreatment of cells with CCK attenuated the association of PP2A with PDE4D5 and the effect of CCK on PP2A association with PDE4D5 was blocked by GF-109203X (1 µM) but not by the Rho kinase inhibitor Y-27632 (1 µM) (Fig. 7). CCK alone had no effect on the association of PP2A with PDE4D5 (data not shown). Treatment of cells with a PMA (1 µM) attenuated forskolin-induced association of PP2A with phosphorylated PDE4D5 (Fig. 7). These results suggest that PKC augments PDE4D5 phosphorylation by inhibiting the interaction of PP2A with PDE4D5, and, thus, PP2A-mediated dephosphorylation. In vitro studies demonstrated phosphorylation of PP2A catalytic subunit by PKC (Fig. 7A).


Figure 7
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  		Fig. 7. PKC-mediated phosphorylation of PP2A and decrease in the association of PP2A with the phosphorylated PDE4D5. A: purified PP2A catalytic subunit was incubated in the absence (lane 1) or presence of purified PKC (5 units) with 0.2 mM [{gamma}-32P]ATP in kinase buffer containing 50 mM Tris·HCl, pH 7.0, 10% glycerol, 1 mM benzamidine, 0.1 phenylmethylsulfonyl fluoride, 14 mM β-mercaptoethanol, 1 µg/ml of phospholipids, and 1 mM MgCl2 for 30 min followed by separation of protein by SDS-PAGE. Radioactive bands corresponding to PP2A catalytic subunit were visualized by autoradiogram of the dried gel. B: gastric muscle cells were incubated with forskolin (10 µM), CCK (1 nM), or PMA for 5 min. The effect of CCK was measured in the absence or presence of GF-109203X (1 µM) or Y-27632 (1 µM). The association of PP2A catalytic subunit with PDE4D5 was measured by immunoblot in PDE4D5 immunoprecipitates. **P < 0.01 significant increase in PP2A-PDE4D5 association.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we have demonstrated that concurrent activation of PKC by contractile agonists can cause augmentation of PKA-induced PDE4D5 phosphorylation and activity in freshly dispersed gastric smooth muscle cells (Fig. 8 ). The effect of PKC is mediated by inhibition of PP2A, which causes increase in the phosphorylation of PDE4D5 by PKA on Ser126. The conclusion that CCK-induced activation of PKC and subsequent inhibition of PP2A is important for augmentation of PDE4D5 phosphorylation and activity leading to decrease in cAMP levels is based on the following evidence: 1) CCK augmented forskolin-induced PDE4D phosphorylation and activity and caused a concurrent decrease in cAMP levels; 2) the increase in PDE4D5 phosphorylation and activity and the decrease in cAMP levels were reversed by the inhibitor of PLC-β or PKC in freshly dispersed cells and in cells expressing G{alpha}q minigene; 3) the selective PP2A inhibitor mimicked the ability of CCK to augment PDE4D5 phosphorylation and activity and inhibit cAMP levels, implying that the effect was mediated by the PKC-dependent inhibition of PP2A; 4) PDE4D5 phosphorylation was selectively dephosphorylated by purified PP2A in vitro; and 5) activation of PKC with CCK attenuated the association of PP2A with phosphorylated PDE4D5. The increase in PDE4D5 phosphorylation and activity and the accompanying decrease in cAMP levels could be mimicked by direct activators of PKC such as PMA. These results suggest that augmentation of PDE4D5 phosphorylation and activity by PKC represents a novel mechanism by which contractile agonists restrain cAMP signaling pathways to rapidly terminate relaxation. Termination of cAMP/PKA signal is also important to restrain PKG signal, because cAMP activates PKA and at higher concentrations cross-activates PKG (19). cAMP via activation of PKA and/or PKG act at various steps in the contractile pathway either to block MLC20 phosphorylation or promote dephosphorylation of MLC20 resulting in muscle relaxation. For example, PKA has been shown to decrease inositol 1,4,5-trisphosphate (IP3) formation, IP3-dependent Ca2+ release, and RhoA-dependent signaling (27, 30). Therefore, rapid and efficient termination of cAMP signaling is essential for apposite signaling by contractile agonists.


Figure 8
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  		Fig. 8. Cross-regulation of cAMP levels by contractile agonists. In gastrointestinal smooth muscle cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific PDE4D5 and stimulation of PDE4D5 activity in a feedback mechanism. Concurrent activation of contractile pathway attenuates cAMP levels by augmenting PKA-mediated PDE4D5 phosphorylation and activity. The pathway involves G{alpha}q-dependent activation of phospholipase C-β (PLC-β) and stimulation of protein kinase C (PKC) resulting in inhibition of protein phosphatase 2A (PP2A) and sustained phosphorylation of PDE4D5.

 
Contractile agonists are shown to be able to modulate intracellular cAMP levels independent of PDE4D5 activation via inhibition of Gs-coupled VIP receptor (VPAC2) internalization by G protein-coupled receptor kinase 2 (GRK2). The mechanism involves phosphorylation of RKIP (Raf kinase inhibitory protein) by PKC and phosphorylation-dependent switching of RKIP from its association with Raf-1 to association with GRK2 and inhibition of GRK2-mediated VPAC2 receptor internalization (15). The results presented here suggest an alternate mechanism through which contractile agonists can decrease cAMP levels by stimulatory phosphorylation of PDE4D5. Such a mechanism of cross-regulation of cAMP by contractile agonists serves to limit the actions of inhibitory transmitters. Thus prior PKC-mediated inhibition of PP2A by contractile agonists would serve to sensitize the cells with augmented PDE4D5 activity to the actions of Gs-coupled relaxant agonists.

The PDE4 family of enzymes exclusively hydrolysis cAMP and provides a dynamic and efficient means of regulating levels. Phosphorylation is the main posttranslational mechanism to regulate PDE4D. A number of kinases including PKA, ERK, and a kinase downstream of PI 3-kinase have been shown to phosphorylate one or more of serine residues of PDE4D long isoforms (11, 14, 20–22, 24, 25). Phosphorylation by PKA has distinct effects on PDE4D3: phosphorylation on Ser54 causes significant increase in cAMP-hydrolyzing activity whereas phosphorylation on Ser13 promotes binding to mAKAP (muscle-selective A-kinase anchoring protein), a scaffolding protein that anchors PDE4D to distinct cellular compartments (4, 14, 20, 21). In contrast to the effect of PKA, the effect of phosphorylation by ERK is cell type specific. Phosphorylation by ERK causes activation of PDE4 in both vascular smooth muscle cells and in FDCP2 myeloid cells but causes inhibition of long PDE4D isoforms in COS-1 and 3t3-F442A fibroblasts and in human embryonic kidney 293 cells (2, 3). The inhibitory phosphorylation of PDE4D3 by ERK at Ser579 can be masked by phosphorylation at an additional site Ser239 by unidentified enzyme downstream of PI 3-kinase (11). Phosphorylation at Ser239 alone, however, had no effect on the activity of PDE4D3, but it switched the effect from inhibition to activation. In this study we have identified a further degree of complexity in the regulation of PDE4D long isoform such as PDE4D5. A novel role for the PKC-PP2A pathway has been defined in the regulation of PDE4D5 activity whereby activation of this pathway led to a phosphorylation-dependent activation of PDE4D5.

PP2A, a family of serine/threonine phosphatases, consists of catalytic and family of regulatory subunits (7, 18). The regulatory subunits determine the substrate specificity, cellular localization, and catalytic activity of the holoenzyme, and the effect of regulatory subunits is modulated by phosphorylation (1, 17, 36). The catalytic subunit is highly conserved. Phosphorylation of the catalytic subunit also appears to regulate the activity. In vitro studies have shown that phosphorylation at Tyr307 by the tyrosine kinase pp6v-Src and by epidermal growth factor and insulin results in inactivation of PP2A (8). This inactivation of PP2A could lead to augmentation of signals though kinase cascades. In addition to phosphorylation on Tyr307, PP2A catalytic subunit can also be phosphorylated in vitro on threonine residue leading to inactivation of the enzyme (9). Consistent with these previous studies on the tyrosine and threonine phosphorylation, our in vitro studies demonstrate direct phosphorylation of PP2A catalytic subunit by PKC and phosphorylation negatively regulates PP2A activity. Thus the phosphorylation-dependent inhibition of PP2A contributes to the increase in PDE4D5 phosphorylation on Ser126 in response to cAMP-elevating agents. The results presented in this study also suggest a mechanism through which contractile agonists can decrease cAMP levels by augmenting PKA-mediated PDE4D5 phosphorylation and activity. The results with PMA suggest that this mechanism would likely be exploited by any stimulus that leads to activation of PKC. The present studies, however, did not identify the specific PKC isoforms(s) responsible for the inhibition of PP2A and augmentation of PDE4D5 phosphorylation and activity. Based on the inhibition by GF-109203X, conventional and/or novel PKC isoforms are likely to be involved in the inhibition of PP1 activity. Further studies are required to identify the PKC isoforms(s) that are involved CCK-induced augmentation of PDE4D5 phosphorylation and activity.

In addition to the modulation of PP2A holoenzyme by regulatory and catalytic subunits, proteins that bind specifically to catalytic subunit play a role in modulation of PP2A catalytic activity (5, 23). Recent studies in smooth muscle have shown that AKAP79 anchors PKA, adenylyl cyclase V/VI, PDE4D5, and PP2A into a molecular scaffold that fosters feedback regulation of cyclase and phosphodiesterase activities by PKA, leading to rapid termination of the cAMP signal in smooth muscle cells (K. S. Murthy, unpublished data). The association of PP2A within the same complex provides a mechanism for rapid dephosphorylation of PDE4D5 and restoration of its activity. Further studies to examine the role of PKC in the association of PP2A with the complex should provide some insight into the regulation of phosphoproteins in intracellular compartments.

In summary, the present studies identified a novel mechanism of cross talk between PKC and cAMP signaling pathways. This underscores a novel means whereby contractile agonists able to activate PKC could decrease cAMP levels and limit signaling by relaxant agonists and optimize the contractile response.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by Grant DK-28300 from the National Institute of Diabetes, and Digestive and Kidney Diseases.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. S. Murthy, Depts. of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298 (e-mail: skarnam{at}vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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