Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

Rap1-B is phosphorylated by protein kinase A in intact human platelets.

Agonists that increase cAMP levels in platelets promote the phosphorylation of a 24 kDa GTP-binding protein that is immunoreactive with a monoclonal antibody (M90) to the H-ras p21 protein. Evidence is presented which indicates that this protein is rap-1b, not rap1-a as previously suggested (Ohmori, T., Kikuchi, A., Yamamoto, K., Kawata, M., Kondo, J. and Takai, Y. (1988) Biochem. Biophys. Res. Commun. 157, 670-676). The amino acid sequence of labeled peptides obtained by proteolytic cleavage of the purified phosphorylated protein was identical with that of rap-1b. Furthermore, a comparison of the kinetics of phosphorylation of synthetic peptides corresponding to the C-terminal region of rap-1a and rap-1b proteins indicated that rap-1b is the preferred substrate for phosphorylation by cAMP-dependent protein kinase.     

Phosphorylation of caldesmon77 by protein kinase C in vitro and in intact human platelets

Caldesmon is a widely distributed calmodulin- and actin-binding protein which occurs in different forms depending on the tissue or cell type under examination. On the basis of molecular weight, caldesmon species can be divided into two classes: caldesmon77 (Mr 70,000-80,000) and caldesmon150 (Mr 140,000-150,000). We have examined the phosphorylation of caldesmon77 by protein kinase C (the Ca2+/phospholipid-dependent enzyme) in vitro and in intact platelets. Caldesmon77, purified from bovine liver, could be phosphorylated by purified rat brain protein kinase C to a level of approximately 1.0 mol of phosphate per mol of caldesmon77 monomer. Two-dimensional tryptic peptide mapping and phosphoamino acid analysis reveals that caldesmon77 is phosphorylated at two major sites exclusively on serine residues. Following treatment of platelets with tumor-promoting phorbol ester, caldesmon77 phosphorylation was elevated 4-fold. Tryptic peptide mapping of phosphorylated platelet caldesmon77 demonstrates that phosphorylation is most significantly enhanced on two peptides which had migration patterns identical with those of the two major phosphopeptides of bovine liver caldesmon77 phosphorylated in vitro. The results of this study indicate that protein kinase C can phosphorylate caldesmon77 in vitro and in intact platelets, suggesting a role for protein kinase C in the regulation of caldesmon77 function or localization.     

Properties of protein kinase C subspecies in human platelets

Protein kinase C (PKC) from human platelets was resolved into two fractions by hydroxyapatite column chromatography. One of the enzymes was indistinguishable from the brain type III PKC having alpha-sequence in its kinetic and immunological properties. The other enzyme was kinetically different from any of the brain PKC subspecies so far isolated, although it resembled the brain type II PKC having beta-sequence. With H1 histone as substrate, this platelet enzyme was not very sensitive to Ca2+, and activated partly by phosphatidylserine plus diacylglycerol or by free arachidonic acid. Both platelet enzymes could phosphorylate the P47 protein in vitro, but the enzyme physiologically responsible for the P47 protein phosphorylation in the activated platelets remains to be identified.     

KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells

Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.     

Activation of protein kinase C by cis- and trans-octadecadienoic acids in intact human platelets and its potentiation by diacylglycerol

Octadecadienoic acids (linoleic acid and linolelaidic acid) and the diacylglycerol, 1-oleoyl-2-acetyl-rac-glycerol (OAG) concentration-dependently induced activation of gel-filtered human platelets, i.e. aggregation and phosphorylation of 20 kDa and 47 kDa peptides. In contrast, octadecenoic acids (oleic and elaidic acid) and octadecanoic (stearic) acid were inactive. Octadecadienoic acid-induced platelet activation was suppressed by the protein kinase C inhibitor, polymyxin B, but not by the cyclooxygenase inhibitor, indomethacin. OAG-induced activation was potentiated by octadecadienoic acids present at non-stimulatory concentrations. Our data suggest that octadecadienoic acids and diacylglycerol synergistically induce platelet activation via protein kinase C. Furthermore, linolelaidic acid may provide a useful experimental tool to study fatty acid regulation of protein kinase C in intact cells.     

Lipoxygenase activity of intact human platelets

The oxygenation by lipoxygenase of different icosaenoic and docosaenoic acids by intact human platelets was studied. The HPLC analysis of the hydroxy compound (s) derived from icosaenoic acids showed that the 12-derivatives predominate. The increase of the fatty acid concentration markedly enhanced their oxygenation except for icosapentaenoic acid. The conversion of this acid into its hydroxy derivative rose in the presence of arachidonic acid, probably through both its cyclo-oxygeuase and lipoxygenase product formation. Since 12-hydroxy-icosaenoic acids are modulators of PGH₂-induced platelet aggregation, we conclude that the interactions between polyunsaturated fatty acids during their oxygenation by platelet lipoxygenase could be relevant to the regulating activity of dietary fatty acids.     

Evidence that cytosolic phospholipase A2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate.

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4-), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4(-)-induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4(-)-induced release of arachidonic acid by a mechanism that is down-regulated by PKC.     

Diglyceride kinase in human platelets

Human platelets contain diglyceride kinase, an enzyme that catalyzes the phosphorylation of diacylglycerol by adenosine 5'-triphosphate to yield phosphatidic acid. The majority of the platelet enzyme is particulate-bound, and membrane fractions of platelet homogenates have a higher specific activity than granule fractions. Both deoxycholate and magnesium are necessary for optimal enzyme activity. The K(m) of the enzyme for adenosine 5'-triphosphate is 1.3 mm, and the apparent K(m) for diacylglycerol is 0.4 mm. The pH optimum is 6.6-6.8 in imidazole-HCl or maleate-NaOH buffer. The enzyme activity of platelets from normal subjects was similar to the activity from patients with renal and hepatic failure.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.     

Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.     

Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.     

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.     

Activators of protein kinase C decrease serotonin transport in human platelets.

Treatment of human platelets with activators of protein kinase C (PKC) for 5-20 min resulted in substantial reductions in the rate of platelet serotonin (5-HT) transport. The mean Vmax observed after 5 min treatment with 1 microM 4-beta-12-tetradecanoylphorbol 13-acetate (beta-TPA) was 66% (n = 16, P = 0.0001) of the control value. 5 min of treatment with 1 microM mezerein reduced uptake to 78% (n = 3, P = 0.01) of control. Both beta-TPA and mezerein had little effect on the Km of transport and had EC50 values of approx. 100 mM when a 20-min treatment period was used. The maximum effects of both were reached at approx. 20 min and could be blocked with staurospine. The beta-TPA effect was stereospecific, as alpha-TPA did not alter platelet 5-HT uptake. Although the PKC activators may have altered transmembrane ion-gradients for Na+ and Cl-, which are co-transported with 5-HT, minimizing ion-gradient changes had little effect on the observed reductions in transport. The PKC activators also had little or no effect on platelet 5-HT release or on the number (Bmax) of 5-HT transporters expressed at the platelet surface. The data indicate that PKC activation may down-regulate the activity of the 5-HT transporter in platelets. Apparently, most of this effect is mediated through mechanisms other than changes in ion-gradients, reductions in the number of available transporters, or increased 5-HT release. The apparent regulation of 5-HT transport by PKC may have important implications in platelet and neuronal functioning.     

Receptor-activated single channels in intact human platelets.

We report "cell-attached" patch clamp studies of intact human platelets which show receptor-activated single channels. Inclusion of ADP in the patch pipette, but not in the bath, resulted in the appearance of inward currents indicative of single channels tightly coupled to the ADP receptors. The channels had a slope conductance of 11 picosiemens at the resting potential. Removal of 1 mM Ca2+ or replacement of chloride by gluconate in the pipette filling solution had little effect on the slope conductance at the resting potential or on the estimated reversed potential. With isotonic BaCl2 in the pipette, ADP evoked single channel currents with a slope conductance of 10 picosiemens. Thus these channels appear to be permeable to monovalent and divalent cations and selective for cations over anions. Addition of 5 mM Ni2+ (which blocks ADP-evoked rapid calcium entry in fura-2-loaded platelets) to the pipette solution blocked ADP-evoked channel activity. These channels may therefore provide an important mechanism for ADP to activate human platelets within a small fraction of a second.     

Selective secretion of protein kinase C isozymes by thrombin-stimulated human platelets

The protein kinase C (PKC) was secreted from thrombin-stimulated human platelets in a time- and dose-dependent manner. The PKC specific inhibitors Ro31-8220 (0.05 microM) and GF 109203X (0.5 microM) totally inhibited the secreted kinase activity. Western blot analysis of the secretory components showed reactivity to PKCalpha, PKCbetaII, and PKCdelta antibodies, but not to PKCbetaI, and p42/44 MAPK, although they were present in lysed platelets. The fractionation of platelets secreted components showed that PKC activity increased in both soluble and microparticle fractions after thrombin treatments. This is the first report demonstrating that activated human platelets selectively secrete protein kinase C isozymes. Protein kinase C secreted by platelets in this unique manner may have an extracellular role in the plasma, and may regulate cellular functions, including remodeling of vascular endothelial cells.     

Phorbol ester induces differential membrane-association of protein kinase C subspecies in human platelets

Human platelets contained proteins which cross-reacted with antisera specific for brain protein kinase C-alpha and -beta. When platelets were incubated with 12-O-tetradecanoylphorbol-13-acetate there was a rapid accumulation of protein kinase C-alpha in the particulate fraction associated with a loss of this subspecies from the soluble fraction. No particulate accumulation or soluble loss of protein kinase C-beta could be detected when platelets were incubated with the phorbol ester.     

Physical and functional interaction between protein kinase C delta and Fyn tyrosine kinase in human platelets

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCdelta and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCdelta on tyrosine and is specific in that other isoforms of PKC, PKCepsilon and lambda, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCdelta does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCdelta and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCdelta are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCdelta activity, did not block translocation of either PKCdelta or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.     

Novel benzofuran inhibitors of human mitogen-activated protein kinase phosphatase-1

Protein tyrosine phosphatases have a central role in the maintenance of normal cellular functionality. For example, PTP1B has been implicated in insulin-resistance, obesity, and neoplasia. Mitogen-activated protein kinase phosphatase-1 (MKP-1 or DUSP1) dephosphorylates and inactivates mitogen-activated protein kinase (MAPK) substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The lack of readily available selective small molecule inhibitors of MKP family members has severely limited interrogation of their biological role. Inspired by a previously identified inhibitor (NSC 357756) of MKP-3, we synthesized seven NSC 357756 congeners, which were evaluated for in vitro inhibition against several protein phosphatases. Remarkably, none displayed potent inhibition against MKP-3, including the desamino NSC 357756 analog NU-154. Interestingly, NU-154 inhibited human PTP1B in vitro with an IC(50) value of 24 +/- 1 microM and showed little inhibition against Cdc25B, MKP-1, and VHR phosphatases. NU-126 [2-((E)-2-(5-cyanobenzofuran-2-yl)vinyl)-1H-indole-6-carbonitrile] inhibited MKP-1 and VHR in vitro but was less active against human MKP-3, Cdc25B, and PTP1B. The inhibition of MKP-1 by NU-126 was independent of redox processes. The benzofuran substructure represents a new potential scaffold for further analog development and provides encouragement that more selective and potent inhibitors of MKP family members may be achievable.