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Am J Physiol Heart Circ Physiol 292: H792-H799, 2007. First published September 29, 2006; doi:10.1152/ajpheart.00225.2006
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Acute modulation of PP2a and troponin I phosphorylation in ventricular myocytes: studies with a novel PP2a peptide inhibitor

Prajwal A. Deshmukh, Bradford C. Blunt, and Polly A. Hofmann

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 2 March 2006 ; accepted in final form 23 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study demonstrates that acute activation with either beta-adrenergic receptor agonists or H2O2 treatment increases protein phosphatase 2a (PP2a) activity in ventricular myocytes. PP2a activation occurs concomitant with an increase in methylation of PP2a, changes in localization of a PP2a targeting subunit PP2aB56{alpha}, and a decrease in phosphorylation of PP2a substrates, such as troponin I (TnI) and ERK in ventricular myocytes. Okadaic acid, a well-established pharmacological inhibitor of PP2a, and the peptide Thr-Pro-Asp-Tyr-Phe-Leu (TPDYFL) were used to block PP2a methylation, localization, and phosphorylations. TPDYFL is a highly conserved sequence of the PP2a catalytic subunit COOH-terminus. Specifically, both okadaic acid and the peptide increased beta-adrenergic-cAMP-dependent phosphorylation of TnI and blocked the beta-adrenergic-cAMP-dependent translocation of PP2aB56{alpha}. TPDYFL, but not a scrambled version of this sequence, blocked H2O2-induced changes in PP2a methylation and TnI dephosphorylation. Okadaic acid produces similar inhibition of H2O2 effects. Thus we propose that the novel peptide TPDYFL acts as an inhibitor of PP2a activity and may be a useful tool to increase our understanding of how PP2a is regulated and the role of PP2a in a variety of physiological and pathological processes. In addition, the present study is consistent with acute beta-adrenergic receptor activation and H2O2 exposure, simultaneously activating kinases and PP2a to work on common substrates, such as TnI. We hypothesize that dual activation of opposing enzymes provides for a tighter regulation of substrate phosphorylations in ventricular myocytes.

beta-adrenergic receptor; hydrogen peroxide; peptide inhibition; protein phosphatase 2a

beta-ADRENERGIC RECEPTOR ACTIVATION increases cAMP and PKA activity in the heart. Myocardial proteins, which are phosphorylated by PKA, include troponin I (TnI), L-type Ca2+ channels, ryanodine receptors, and phospholamban. The notable end effects of PKA-dependent phosphorylations include a positive inotropic response and increase in the rate of relaxation of the heart. beta-Adrenergic receptors may also simultaneously activate the serine/threonine protein phosphatase 2a (PP2a). Yeast two-hybrid screening has demonstrated that at least one adenylate cyclase isoform and PP2a are associated (6), and cAMP increases methylation of PP2a in frog eggs (10). Methylation of PP2a increases PP2a activity localized to substrates (3, 29). Furthermore, beta2-adrenergic receptors appear to activate a serine/threonine phosphatase to reduce adrenergic-dependent increases in contractility in ventricular myocytes (16). These data taken together are consistent with the hypothesis that a beta-adrenergic-dependent increase in PP2a activity attenuates PKA-induced phosphorylations and subsequent effects. Thus one goal of the present study was to determine whether beta-adrenergic-cAMP stimulation increases PP2a activity and whether this then leads to a decrease in cAMP-dependent phosphorylation of TnI. These observations are of clinical interest in that if simultaneous activation of PP2a and PKA does occur, then pharmacological inhibition of PP2a may allow for greater-than-normal beta-adrenergic inotropic support of acutely failing hearts.

Acute H2O2 treatment has been shown to activate PKC-beta and, subsequently, p90 ribosomal S6 kinase (p90rsk) (14). p90rsk phosphorylates Ser23/24 on TnI in cardiomyocytes (14). Acute H2O2 treatment also increases PP2a enzymatic activity toward ERK in ventricular myocytes (25). Thus we hypothesize that H2O2-dependent increases in PP2a may oppose phosphorylation events and effects of H2O2-induced kinase activations in ventricular myocytes. As such, another goal of the present study was to determine whether H2O2-stimulated PP2a activity decreases phosphorylation of TnI.

The final goal of the present study was to test a novel peptide inhibitor of PP2a. Short-term inhibition of PP2a activity in the laboratory has relied on an array of naturally occurring inhibitors such as okadaic acid (8, 12, 19). However, specific inhibition can also occur through the use of a peptide sequence that competitively interferes with a step in the activation of an enzyme (2, 7, 21, 22, 32). This led us to carefully consider what is known about PP2a activation. In brief, the holoenzyme of PP2a contains a scaffolding (PP2aA), catalytic (PP2aC), and regulatory/targeting (PP2aB) subunit (reviewed in Refs. 15, 27, and 34). PP2aA and PP2aC form a constitutive diamer (PP2aAC), and an association of one of the highly diverse isoforms of PP2aB to PP2aAC confers substrate specificity and subcellular localization of the active enzyme. Posttranslational modifications of the PP2a catalytic subunit occur through phosphorylation of Tyr307 (4) and reversible methylation of Leu309 at the COOH-terminus of PP2aC (9, 17, 18, 20). Methylation of Leu is thought to promote PP2aB binding to PP2aAC and, as such, alter the localized PP2a activity (3, 29). Deletions or mutations in Tyr307 also influence PP2aB binding to PP2aAC and holoenzyme subcellular localization (13). The COOH-terminal end sequence of PP2aC containing Tyr307 and Leu309, Thr-Pro-Asp-Tyr-Phe-Leu (TPDYFL), is conserved in all known sequences of PP2aC from yeast to humans (1, 33). Thus the final goal of the present study was to test the ability of an exogenous PP2a COOH-terminal-derived peptide, TPDYFL, to inhibit PP2a-dependent effects in ventricular myocytes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Cell isolation, agonist treatment, and peptide incorporation into ventricular myocytes. Rats were handled in accordance with the guidelines of the Animal Care and Use Committee of the University of Tennessee Health Science Center. Ventricular myocytes were enzymatically isolated from adult female Wistar rats as previously described (23).

For determination of beta-adrenergic effects on PP2a enzymatic activity, cells were treated for 2 min with vehicle (control); 1 x 10–4 M salbutamol, a beta2-adrenergic receptor agonist; or salbutamol plus 2.5 x 10–6 M ICI-118551, a beta2-adrenergic antagonist. In other experiments cells were treated with the general beta1/beta2-adrenergic receptor agonist isoproterenol at 100 nM for 2 min.

Peptides were incorporated into myocytes using a solution containing beta-escin and the protocol of Dobson and colleagues (28). The peptides (see Table 1; Genemed Synthesis, San Francisco, CA) were in the permeabilization solution at a concentration of 0.15 µg/µl unless otherwise noted. Following peptide incorporation, the cells were resealed (28) and shown to be Ca2+ tolerant in a 1 mM Ca2+ Ringer solution. Subsequent treatment protocols for peptide-incorporated cells are presented in Table 1.


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  		Table 1. Peptide and scrambled peptide experimental treatment groups

 
PP2a phosphatase activity assay. Freshly isolated ventricular myocytes were treated with beta-adrenergic agonists/antagonists, and cells were lysed with a solution containing 0.1% Nonidet P-40 and (in mM) 5 EDTA, 5 DTT, 20 Tris (pH 7.4), 150 NaCl, and 1 vanadate. The supernatant containing PP2a was collected and PP2a activity determined by the rate of Pi release upon addition of a prephosphorylated PP2a substrate (Arg-Arg-Ala-phosphorylated Thr-Val-Ala) in the presence and absence of 5 nM okadaic acid. Assay conditions were followed as specified in the instructions provided with the substrate (Cat. No. C2460; Promega, Madison, WI).

To determine the effect of acute H2O2 treatment on PP2a activity in myocyte fractions, cells were treated with vehicle or 100 µM H2O2 for 5 min, washed, and mechanically homogenized in a solution containing 0.1% Triton X-100 and (in mM) 2 EDTA, 25 Tris (pH 7.4), 150 KCl, 1 vanadate, and 1 NaF. Following centrifugation at 1,000 g for 2 min, the supernatant was retained and designated as the membrane-cytosolic fraction, whereas the pellet was redissolved in the above buffer and designated as the myofilament-particulate fraction. Aliquots of both of these fractions were combined with a fluorescent, prephosphorylated peptide that, upon dephosphorylation, increases its fluorescence intensity (IQ Assay; Pierce Chemical, Rockford, IL). This assay is not affected by baseline Pi values that can be high and/or variable in myocardial preparations. Fractions from the cells plus the fluorescent peptide plus either no okadaic acid or 10 nM okadaic acid were combined for 1 h with constant agitation at room temperature. Fluorescence intensity at 560-excitation/590-emission wavelengths was then determined for the membrane-cytosolic fraction. Assays using the myofilament-particulate fraction underwent an additional centrifugation at 2,000 g for 30 s, and the resulting translucent supernatant was used to determine changes in fluorescence intensity.

To establish the effect of the inhibitory peptide on PP2a activity in vitro, freshly isolated ventricular myocytes were lysed with a solution containing 0.5% Triton X-100, 5 mM EDTA, and 5 mM DTT. The supernatant containing endogenous PP2a was added to prephosphorylated PP2a substrate in the presence and absence of the test peptides (Table 1). PP2a activity was determined by the rate of Pi release in the presence and absence of 5 nM okadaic acid.

Fractionation and determination of PP2aB56{alpha}. Following treatment, cells were triturated and placed on ice for 5 min in a buffer containing 0.05% digitonin and (in mM) 20 HEPES (pH 7.4), 10 KCl, 1.5 MgCl2, and 5 DTT. This treatment allows for release of the cytosolic component of cells without solubalizing membrane-associated proteins (30, 35). The supernatant collected following centrifugation at 12,000 g for 5 min was designated the cytosolic fraction, whereas the pellet was redissolved and designated as the particulate. Aliquots were combined with an equal volume of urea containing electrophoretic sample buffer, separated by gel electrophoresis, and transferred to PVDF membranes. For Western blot analyses, the PP2aB56{alpha} antibody (Cat No. SC-6116; Santa Cruz Biotechnology, Santa Cruz, CA) was used at a dilution of 1:500. Coomassie-stained gels were also obtained, and densitometry showed minor variation in protein load but an overall equality in loads within a given isolation.

Western blot determination of phospho-TnI, ERK, and demethylated PP2aC. The extent of phosphorylated TnI, phosphorylated ERK, or demethylated PP2aC was determined in whole cells. After indicated drug treatments, the cells were combined with a Laemmli sample buffer containing 50 mM NaF, 100 µM sodium orthovanadate, 5 mM EDTA, and 5 mM DTT. Western blot protocols were carried out as previously described (23–25). The phosphorylated TnI antibody, phosphorylated ERK antibody, and demethylated PP2a antibody (Cat Nos. 4004, 9101, and 4957, respectively; Cell Signaling Technology, Beverly, MA) were used at a dilution of 1:500. Coomassie-stained gels were also obtained.

Statistics. Values are reported as means ± SE. Statistical significance was taken as P ≤ 0.05. ANOVA was followed by a Tukey honestly significant difference or least significant difference multiple comparison. For studies in which data were normalized to paired vehicle-treated cells, a two-way ANOVA using complete block design without replication and appropriate post hoc tests were completed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
beta-Adrenergic-cAMP activation and PP2a. Acute beta2-adrenergic receptor activation increased total PP2a enzymatic activity in ventricular myocytes (Fig. 1). beta-Adrenergic-mediated cAMP-PKA activation in ventricular myocytes is well known to cause phosphorylation of TnI (reviewed in Ref. 26; Fig. 2). Incorporation of the exogenous PP2a COOH-terminal-derived peptide into ventricular myocytes allowed for a greater increase in cAMP-induced phosphorylation of TnI (Fig. 2). This suggests the exogenous peptide acts to inhibit PP2a activity in vivo to allow for unopposed PKA phosphorylation of TnI.


Figure 1
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  		Fig. 1. Total cell protein phosphatase 2a (PP2a) activity in ventricular myocytes treated with vehicle (Control); the beta2-adrenergic receptor agonist salbutamol, 1 x 10–4 M for 2 min; or a beta2-adrenergic receptor antagonist ICI-118551, 2.5 x 10–6 M, plus salbutamol. Following stimulation, myocytes were lysed, the PP2a containing supernatant was added to an exogenous prephosphorylated PP2a substrate, and the rate of Pi release was determined colorimetrically. *P < 0.05 compared with Control. Data are means ± SE for 4 isolations.

 

Figure 2
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  		Fig. 2. A typical Western blot, corresponding Coomassie-stained gel, and cumulative analysis of the extent of phosphorylation of troponin I (TnI) upon dibutyryl cAMP (cAMP) or vehicle treatment (Con) of ventricular myocytes. Before cAMP exposure, cells underwent a permeabilization procedure in the presence (Peptide) and absence (No Peptide) of a PP2a COOH-terminal-derived peptide and were resealed. *P < 0.05 compared with corresponding Con. #P < 0.05 compared with cAMP, No Peptide. Data are means ± SE for 4 isolations.

 
To test whether the peptide or a scrambled form of the peptide inhibits crudely purified PP2aC enzymatic activity in vitro, soluble PP2a from ventricular myocyte lysates was combined with the peptides and with an exogenous, prephosphorylated substrate specific to PP2a. An increase in PP2a-dependent substrate release of Pi occurs with an increase in assay duration (Fig. 3, No Peptide). However, neither the exogenous PP2a COOH-terminal peptide nor the scrambled version of the peptide influenced the in vitro PP2a catalytic ability (Fig. 3). Peptides were tested at two concentrations with the lower concentration being that used in the in vivo studies (Figs. 2, 4, GoGo7, and 8).


Figure 3
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  		Fig. 3. Cumulative result of Pi release from an exogenous phosphorylated substrate of PP2a by ventricular myocyte PP2aC in the presence of no peptide, the PP2a COOH-terminal-derived peptide of PP2aC, or a scrambled version of the peptide. Two different concentrations of the peptides were used, and Pi release was assayed at 15 and 30 min after addition of the exogenous substrate of PP2a. Increasing assay duration significantly increased the amount of Pi release in all cases. Neither peptide at a high or low concentration significantly altered PP2a-dependent Pi release. Data are presented relative to the amount of Pi released at 15 min in samples with no peptide present. Data are means ± SE for 3 isolations.

 

Figure 4
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  		Fig. 4. Typical Western blots, corresponding Coomassie-stained gel, and cumulative analysis of basal phosphorylation of ERK in ventricular myocytes. Cells underwent a permeabilization procedure in the presence of the PP2aC peptide (Peptide), a scrambled version of that peptide (Scramble), or no peptide before phospho-ERK determination. *P < 0.05 compared with No Peptide. No significant change in total ERK was observed between groups (data not shown). Data are means ± SE for 4 isolations.

 

Figure 5
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  		Fig. 5. Typical Western blots, corresponding Coomassie-stained gel, and cumulative analysis of PP2aB56{alpha} content in the cytosolic (cyto) and particulate (part) fractions of ventricular myocytes stimulated with vehicle (Control), 100 nM isoproterenol for 3 min (Iso), or 0.5 mM dibutyryl cAMP for 30 min (cAMP). *P < 0.05 compared with corresponding Control. Data are means ± SE for 3 isolations.

 

Figure 6
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  		Fig. 6. Typical Western blots, corresponding Coomassie-stained gel, and cumulative analysis of PP2aB56{alpha} content in the cytosolic fraction of ventricular myocytes pretreated with vehicle (No Oka) or 10 nM okadaic acid (10 nM Oka) followed by vehicle (Con) or 0.5 mM dibutyryl cAMP (cAMP) exposure. Oka pretreatment was for 30 min followed by 30 min vehicle or cAMP exposure. Data are normalized to the amount of PP2aB56{alpha} in the cytosolic fraction of myocytes not pretreated with Oka or cAMP. *P < 0.05 compared with corresponding Con; #P < 0.05 compared with cAMP, No Oka. Data are means ± SE for 3 isolations.

 

Figure 7
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  		Fig. 7. Typical Western blots, corresponding Coomassie-stained gel, and cumulative analysis of PP2aB56{alpha} content in the cytosolic fraction of ventricular myocytes treated with dibutyryl cAMP. Before cAMP exposure, cells were permeabilized in the presence of the PP2aC peptide (Peptide), a scrambled version of that peptide (Scramble), no peptide, or not permeabilized (Intact Cells). Data are normalized to the amount of PP2aB56{alpha} in the cytosolic fraction of myocytes not treated with cAMP for each group. *P < 0.05 compared with non-cAMP-treated cells from that same group. Data are means ± SE for 5 isolations.

 

Figure 8
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  		Fig. 8. PP2a activity in fractions of ventricular myocytes that, before fractionation, were treated with vehicle (Con or C) or 100 µm H2O2 (H2O2 or H) for 5 min. Inset: representative Coomassie-stained gel of the samples utilized for determination of PP2a activity. Following vehicle or H2O2 treatment, myocytes were homogenized in a Triton-containing solution and centrifuged to obtain the Triton-soluble, membrane-cytosolic (Mem-Cyto) supernatant and the Triton-insoluble, myofilament-particulate (Myo-Part) fraction. Fractions were then incubated with a fluorescent, prephosphorylated peptide that increases in fluorescence intensity upon dephosphorylation. Data are expressed as the difference in fluorescence between samples containing no okadaic acid and samples containing 10 nM okadaic acid, a concentration selective for PP2a inhibition. *P < 0.05 compared with corresponding Control. Data are means ± SE for 7 isolations.

 
To determine whether the PP2a COOH-terminal peptide effects the phosphorylation status of another known PP2a substrate in vivo, the phosphorylation of ERK2 was examined. Incorporation of the exogenous PP2a COOH-terminal-derived peptide into ventricular myocytes caused a modest increase in basal phosphorylation of ERK (Fig. 4). Incorporation of the scrambled sequence of this peptide had no effect on the extent of basal ERK phosphorylation. This is consistent with the exogenous COOH-terminal peptide of PP2a acting as a PP2a inhibitor.

The subcellular localization of PP2aB56{alpha} was determined in cells treated with isoproterenol, a beta-adrenergic receptor agonist, or dibutyryl cAMP. Both isoproterenol and dibutyryl cAMP increased the cytoplasmic concentration of PP2aB56{alpha}, and a decrease in PP2aB56{alpha} in the particulate was seen with dibutyryl cAMP treatment (Fig. 5). The lack of effect of isoproterenol on the particulate may be a function of the low PP2aB56{alpha} signal of the pellet. PP2aB56{alpha} concentration appeared low in the particulate since it was necessary to dilute this fraction, composed primarily of contractile proteins, so that samples could be run on SDS-PAGE.

In a separate series of experiments, cells were pretreated for 30 min with 10 nM okadaic acid before dibutyryl cAMP treatment and the cytosolic concentration of PP2aB56{alpha} was determined. Pretreatment with okadaic acid attenuated the cAMP-induced increase in cytosolic PP2aB56{alpha} (Fig. 6). Experiments using the PP2a COOH-terminal-derived peptide were also completed. Dibutyryl cAMP caused a translocation of PP2aB56{alpha} to the cytosolic fraction of ventricular myocytes (Fig. 7) in both freshly isolated, intact cells and in cells that underwent the permeabilization-resealing procedure (No Peptide). Translocation to the cytosolic fraction was fully blocked by preincorporation of the exogenous PP2a COOH-terminal peptide, whereas incorporation of the scrambled version of the PP2a peptide did not alter the cAMP-induced translocation of PP2aB56{alpha} (Fig. 7).

Effect of peptides on in vivo H2O2-induced changes in PP2a activity. Previously, we have shown that acute H2O2 treatment, through p38 MAPK activation, increases PP2a association and activity associated with ERK in cardiomyocytes (25). In the present study, acute H2O2 treatment increased PP2a activity associated with the myofilament-particulate fraction and decreased PP2a activity in the Triton-soluble membrane-cytosolic fraction (Fig. 8). Furthermore, H2O2 treatment decreased the extent of demethylated PP2a in cells (Fig. 9). A decrease in demethylated PP2aC is expected to increase localized PP2a activity since demethylated PP2aC has reduced the PP2aB binding and targeting ability (3, 29). Consistent with the H2O2-induced decrease in demethylated PP2a and increase in PP2a activity in myofilaments, acute H2O2 treatment decreased phosphorylation of TnI in ventricular myocytes, and this decrease was sensitive to okadaic acid (Fig. 10). The exogenous PP2a COOH-terminal-derived peptide blocked the H2O2-dependent decreases in demethylated PP2a and phosphorylation of TnI, whereas the scrambled version of the peptide did not (Fig. 9).


Figure 9
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  		Fig. 9. Typical Western blots, corresponding Coomassie-stained gel, and cumulative analysis of demethylated PP2aC and phosphorylated TnI upon H2O2 or vehicle (Con) treatment. Before H2O2 exposure, cells underwent a permeabilization procedure in the presence of the PP2aC peptide (Peptide), a scrambled version of that peptide (Scramble), or no peptide and were resealed. *P < 0.05 compared with corresponding Con. #P = 0.08 compared with Con, No Peptide. Data are means ± SE for 5 isolations.

 

Figure 10
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  		Fig. 10. Typical Western blots, corresponding Coomassie-stained gel, and cumulative analysis of phosphorylated TnI (P-TnI) in ventricular myocytes vehicle pretreated (No Oka) or pretreated with 10 nM okadaic acid and stimulated with H2O2 or vehicle (Con). Okadaic pretreatment was for 1 h, 100 nM Iso treatment was for 2 min, and 100 µM H2O2 treatment was for 15 min. P-TnI density values were adjusted for slight variations in protein load and normalized to corresponding Con. *P < 0.05 compared with corresponding Con. Data are means ± SE for 4 isolations.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that beta-adrenergic receptor stimulation leads to a redistribution of PP2aB56{alpha} and to an increase in total cell PP2a activity in ventricular myocytes. These changes reduce the extent of phosphorylation of TnI that was brought about by beta-adrenergic-PKA activation (Fig. 11). The present study also suggests an H2O2-dependent increase in PP2a activity occurs concomitant with a change in the methylation of the PP2aC subunit and a dephosphorylation of TnI (Figs. 8–10). We hypothesize that this would antagonize phosphorylation of TnI brought about by H2O2-p90rsk activation (14). Finally, the present study investigates the ability of a PP2a COOH-terminal-derived peptide to inhibit endogenous PP2a activity. The exogenous peptide does not appear to be a direct inhibitor of PP2a catalytic activity, but rather decreases endogenous methylation and/or PP2aB association and PP2a holoenzyme translocation. As such, the peptide derived from the COOH-terminal sequence of PP2a, TPDYFL, appears to be a promising tool to better understand the role of PP2a in various cell processes.


Figure 11
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  		Fig. 11. Diagram of hypothesized pathways.

 
Previous studies have demonstrated an association of PP2a and adenylyl cyclase VIII in neuronal tissue (6), and cAMP increases methylation of PP2aC in frog eggs (10). In myocardium, pharmacological inhibition of serine/threonine phosphatases increases the beta2-adrenergic but not the beta1-adrenergic positive inotropic effect (16). This suggests a beta2-adrenergic dual activation of a phosphatase and PKA, such that the phosphatase attenuates the PKA-dependent phosphorylation of L-type Ca2+ channels. The present study demonstrates that acute activation of beta-adrenergic receptor stimulation does increase PP2a enzymatic activity in intact ventricular myocytes (Fig. 1), and the mechanism to do so may involve a beta-adrenergic-cAMP-dependent change in PP2aB56{alpha} subcellular localization (Fig. 5). An observation of stimulation-dependent localization raises the possibility that the increase in PP2a activity in subcellular compartments may be even higher than the measured beta-adrenergic-induced increases in PP2a activity of whole cells (Fig. 1). Consistent with a beta-adrenergic-PP2a activation pathway are the observations that the inhibition of PP2a activation, using okadaic acid or an exogenous peptide with a short sequence of the COOH-terminal of PP2a, enhances beta-adrenergic-induced phosphorylation of TnI and inhibits beta-adrenergic-cAMP-dependent PP2aB56{alpha} translocation (Figs. 2, 6, and 7). The functional implication of a beta-adrenergic-PP2a pathway is that the beta-adrenergic-PKA-induced increase in myocardial contractility would be attenuated by coactivation of PP2a. As such, in the clinical setting, it is possible that greater-than-normal beta-adrenergic support of the acutely failing heart could occur if beta-adrenergic activation of PP2a were inhibited.

Some studies have demonstrated that H2O2 treatment reduces PP2a activity through a glutathionylation of PP2a (11, 31, 35). However, other studies have shown that H2O2 increases PP2a activity associated with specific PP2a substrates in cells (5, 25). This raises the possibility that localization of PP2a may be an important factor in determining activity against a specific substrate. The present study is consistent with acute, low-dose H2O2 exposure increasing the activity of PP2a against TnI and causing an overall decrease in PP2a activity associated with the membrane and cytosolic compartments. This is based on the observations that 1) H2O2 decreases PP2a activity associated with the Triton-soluble, membrane-cytosolic cellular fraction, 2) H2O2 increases PP2a activity associated with the Triton-insoluble myofilament-particulate cellular fraction, 3) H2O2 decreases the extent of demethylated PP2aC in the whole cell, 4) H2O2 decreases the phosphorylation of TnI in a PP2a-dependent manner, and 5) H2O2-dependent effects on methylation and phosphorylation are blocked by the exogenous PP2a COOH-terminal-derived peptide but not the scrambled version of this peptide. We hypothesize that other PP2a substrates, such as those involved in cell survival, may also be altered by H2O2-induced activation of PP2a to influence when and how reactive oxygen species either improve survival or are proapoptotic in cardiac myocytes (25).

The six amino acid COOH-terminal peptide utilized in the present study contains both the leucine methylation site and the tyrosine phosphorylation site of PP2aC. Thus this peptide could act as a competitive inhibitor of endogenous PP2aC for either of these events. Our studies demonstrate the exogenous peptide causes a trend toward an increase in demethylation of endogenous PP2a (i.e., it inhibits methylation; see Fig. 9 Peptide, Control vs. No Peptide, Control) and blocks the H2O2-induced decrease in demethylation of PP2a in ventricular myocytes (Fig. 9). These observations are consistent with the exogenous peptide being an inhibitor for endogenous PP2aC methylation. However, it should be noted that in vitro studies demonstrate that PP2a COOH-terminal peptides of a length of 4, 8, or 10 amino acids do not inhibit PP2aC methylation (18, 36). One explanation of an in vitro lack of effect on methylation versus in vivo inhibition of methylation by a COOH-terminal-derived peptide is that the in vitro conditions lack an important modulatory event/molecule needed for methylation. Our studies are consistent with that hypothesis in that, in an in vitro PP2a enzymatic assay, the peptide had no effect on PP2a activity (Fig. 3).

The ability of the exogenous PP2a COOH-terminal peptide to block PP2aB56{alpha} translocation (Fig. 7) is consistent with the observed inhibition of PP2aC methylation. Past work has demonstrated that methylation of PP2aC is needed for PP2aB binding and translocation of the holoenzyme PP2a (3, 29). Decreased translocation of the holoenzyme PP2a is consistent with increased TnI and ERK phosphorylation (Figs. 2 and 4). It should also be noted that the effects of the peptide-dependent inhibition of PP2a are consistent with past observations made using okadaic acid. Okadaic acid increases beta-adrenergic-cAMP-induced phosphorylation of TnI (23). Furthermore, a near-identical increase in basal phosphorylation of ERK is seen with okadaic acid (25) and the PP2a COOH-terminal peptide (Fig. 4). Thus incorporation of an exogenous peptide made up of the final six amino acids of PP2aC may act as an inhibitor of endogenous PP2aC methylation in vivo. This, in turn, decreases the amount of PP2aB associated with PP2aAC and reduces the targeting/localization of the holoenzyme to PP2a substrates.

Pharmacological inhibitors of PP2a, such as okadaic acid, have provided us with an inexpensive and relatively selective way to test for the involvement of PP2a. Knockout and mutational studies of PP2a have provided insight into the developmental role of PP2a and PP2a functional effects. The PP2aC COOH-terminal-derived peptide TPDYFL may prove to be an equally valuable tool in understanding the mechanisms by which PP2a is modulated and the functional significance of PP2a in various cell types. In the present application, the PP2a peptide inhibitor was useful in uncovering activations of PP2a that counter the effect of kinase activations to modulate TnI phosphorylation status in ventricular myocytes (Fig. 11).


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This study was supported by National Heart, Lung, and Blood Institute Grant HL-48839 (to P. A. Hofmann).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Hofmann, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: phofmann{at}physio1.utmem.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.


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 REFERENCES
 

  1. Arino J, Woon CW, Brautigan DL, Miller TB Jr, Johnson GL. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes. Proc Natl Acad Sci USA 85: 4252–4256, 1988.[Abstract/Free Full Text]
  2. Boehning D, van Rossum DB, Patterson RL, Snyder SH. A peptide inhibitor of cytochrome c/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and extrinsic cell death pathways. Proc Natl Acad Sci USA 102: 1466–1471, 2005.[Abstract/Free Full Text]
  3. Bryant JC, Westphal RS, Wadzinski BE. Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit. Biochem J 339: 241–246, 1999.
  4. Chen J, Martin BL, Brautigan DL. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257: 1261–1264, 1992.[Abstract/Free Full Text]
  5. Cicchillitti L, Fasanaro P, Biglioli P, Capogrossi MC, Martelli F. Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130. J Biol Chem 278: 19509–19517, 2003.[Abstract/Free Full Text]
  6. Crossthwaite AJ, Ciruela A, Rayner TF, Cooper DM. A direct interaction between the N terminus of adenylyl cyclase AC8 and the catalytic subunit of protein phosphatase 2A. Mol Pharmacol 69: 608–617, 2006.[Abstract/Free Full Text]
  7. Dorn GW 2nd, Mochly-Rosen D. Intracellular transport mechanisms of signal transducers. Annu Rev Physiol 64: 407–429, 2002.[CrossRef][ISI][Medline]
  8. Dounay AB, Forsyth CJ. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. Curr Med Chem 9: 1939–1980, 2002.[ISI][Medline]
  9. Favre B, Zolnierowicz S, Turowski P, Hemmings BA. The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo. J Biol Chem 269: 16311–16317, 1994.[Abstract/Free Full Text]
  10. Floer M, Stock J. Carboxyl methylation of protein phosphatase 2A from Xenopus eggs is stimulated by cAMP and inhibited by okadaic acid. Biochem Biophys Res Commun 198: 372–379, 1994.[CrossRef][ISI][Medline]
  11. Foley TD, Armstrong JJ, Kupchak BR. Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain. Biochem Biophys Res Commun 315: 568–574, 2004.[CrossRef][ISI][Medline]
  12. Gehringer MM. Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response. FEBS Lett 557: 1–8, 2004.[CrossRef][ISI][Medline]
  13. Gentry MS, Li Y, Wei H, Syed FF, Patel SH, Hallberg RL, Pallas DC. A novel assay for protein phosphatase 2A (PP2A) complexes in vivo reveals differential effects of covalent modifications on different Saccharomyces cerevisiae PP2A heterotrimers. Eukaryot Cell 4: 1029–1040, 2005.[Abstract/Free Full Text]
  14. Itoh S, Ding B, Bains CP, Wang N, Takeishi Y, Jalili T, King GL, Walsh RA, Yan C, Abe J. Role of p90 ribosomal S6 kinase (p90RSK) in reactive oxygen species and protein kinase C beta (PKC-beta)-mediated cardiac troponin I phosphorylation. J Biol Chem 280: 24135–24142, 2005.[Abstract/Free Full Text]
  15. Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353: 417–439, 2001.[CrossRef][ISI][Medline]
  16. Kuschel M, Zhou YY, Cheng H, Zhang SJ, Chen Y, Lakatta EG, Xiao RP. Gi protein-mediated functional compartmentalization of cardiac beta2-adrenergic signaling. J Biol Chem 274: 22048–22052, 1999.[Abstract/Free Full Text]
  17. Lee J, Stock J. Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase. J Biol Chem 268: 19192–19195, 1993.[Abstract/Free Full Text]
  18. Lee J, Chen Y, Tolstykh T, Stock J. A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc Natl Acad Sci USA 93: 6043–6047, 1996.[Abstract/Free Full Text]
  19. Lewy DS, Gauss CM, Soenen DR, Boger DL. Fostriecin: chemistry and biology. Curr Med Chem 9: 2005–2032, 2002.[ISI][Medline]
  20. Li M, Damuni Z. Okadaic acid and microcystin-LR directly inhibit the methylation of protein phosphatase 2A by its specific methyltransferase. Biochem Biophys Res Commun 202: 1023–1030, 1994.[CrossRef][ISI][Medline]
  21. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem 270: 14255–14258, 1995.[Abstract/Free Full Text]
  22. Liu KY, Timmons S, Lin YZ, Hawiger J. Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs. Proc Natl Acad Sci USA 93: 11819–11824, 1996.[Abstract/Free Full Text]
  23. Liu Q, Hofmann PA. Antiadrenergic effects of adenosine A1 receptor-mediated protein phosphatase 2a activation in the heart. Am J Physiol Heart Circ Physiol 283: H1314–H1321, 2002.[Abstract/Free Full Text]
  24. Liu Q, Hofmann PA. Modulation of protein phosphatase 2a by adenosine A1 receptors in cardiomyocytes: role for p38 MAPK. Am J Physiol Heart Circ Physiol 285: H97–H103, 2003.[Abstract/Free Full Text]
  25. Liu Q, Hofmann PA. Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes. Am J Physiol Heart Circ Physiol 286: H2204–H2212, 2004.[Abstract/Free Full Text]
  26. Metzger JM, Westfall MV. Covalent and noncovalent modification of thin filament action: the essential role of troponin in cardiac muscle regulation. Circ Res 94: 146–158, 2004.[Abstract/Free Full Text]
  27. Millward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 24: 186–191, 1999.[CrossRef][ISI][Medline]
  28. Miyazaki K, Komatsu S, Ikebe M, Fenton RA, Dobson JG Jr. Protein kinase C{epsilon} and the antiadrenergic action of adenosine in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 287: H1721–H1729, 2004.[Abstract/Free Full Text]
  29. Ogris E, Gibson DM, Pallas DC. Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. Oncogene 15: 911–917, 1997.[CrossRef][ISI][Medline]
  30. Pelech SL, Meier KE, Krebs EG. Rapid microassay for protein kinase C translocation in Swiss 3T3 cells. Biochemistry 25: 8348–8353, 1986.[CrossRef][Medline]
  31. Rao RK, Clayton LW. Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation. Biochem Biophys Res Commun 293: 610–616, 2002.[CrossRef][ISI][Medline]
  32. Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD, Westbrook GL. Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 368: 853–856, 1994.[CrossRef][Medline]
  33. Sneddon AA, Cohen PT, Stark MJ. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. EMBO J 9: 4339–4346, 1990.[ISI][Medline]
  34. Sontag E. Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal 13: 7–16, 2001.[CrossRef][ISI][Medline]
  35. Whisler RL, Goyette MA, Grants IS, Newhouse YG. Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells. Arch Biochem Biophys 319: 23–35, 1995.[CrossRef][ISI][Medline]
  36. Xie H, Clarke S. Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain. J Biol Chem 269: 1981–1984, 1994.[Abstract/Free Full Text]



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