Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein.

The hepatitis C virus E1 and E2 envelope proteins are targeted to the endoplasmic reticulum, but instead of being secreted, they are retained in a pre-Golgi compartment, at least partly in a misfolded state. Since secretory proteins which are retained in the endoplasmic reticulum frequently can activate the transcription of intraluminal chaperone proteins, we measured the effect of the E1 and E2 proteins on the promoters of two such chaperones, GRP78 (BiP) and GRP94. We found that E2 but not E1 protein activates these two promoters, as assayed by a reporter gene system. Furthermore, E2 but not E1 protein induces the synthesis of GRP78 from the endogenous cellular gene. We also found that E2 but not E1 protein expressed in mammalian cells is bound tightly to GRP78. This association may explain the ability of E2 protein to activate transcription, since GRP78 has been postulated to be a sensor of stress in the endoplasmic reticulum. Since overexpression of GRP78 has been shown to decrease the sensitivity of cells to killing by cytotoxic T lymphocytes and to increase tumorigenicity and resistance to antitumor drugs, this activity of E2 protein may be involved in the pathogenesis of hepatitis C virus-induced diseases.

Mutational analysis of the autoinhibitory domain of calmodulin kinase II.

Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.

The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes.

The ATP.Mg-dependent type-1 protein phosphatase and its activating factor (protein kinase FA) were identified to exist in brain synaptosome. The inactive protein phosphatase was found to exist in the synaptosomal cytosol whereas its activating factor (protein kinase FA) was present in the synaptosomal membrane, indicating that the inactive protein phosphatase and its activating factor FA are localized in two separate subcellular compartments. The membrane-bound FA was found to exist in two forms; approximately 75% of FA is inactive and trypsin-resistant, whereas 25% of FA is active and trypsin-labile. When membranes were incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be activated and sequestered to become the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, the results provide initial evidence that the activation-sequestration of membrane-bound protein kinase FA may represent one mode of control modulating the activity of protein kinase FA and thereby to activate protein phosphatase in brain synaptosome, representing an efficient regulatory mechanism for regulating neurotransmission in the central nervous system.

Cyclic phosphorylation-dephosphorylation of rhodopsin in retina by protein kinase FA (the activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) was identified to exist in bovine retina. Furthermore, rhodopsin, the visual light pigment associated with rod outer segments in retina, could be well phosphorylated by kinase FA to about 0.9 mol of phosphates per mol of protein. Moreover, more than 90% of the phosphates in [32P]-rhodopsin could be completely removed by ATP.Mg-dependent protein phosphatase and the rhodopsin phosphatase activity was strictly kinase FA-dependent. Taken together, the results provide initial evidence that a cyclic phosphorylation-dephosphorylation of rhodopsin can be controlled by the retina-associated protein kinase FA, representing an efficient cyclic cascade mechanism possibly involved in the rapid regulation of rhodopsin function in retina.

Expression and characterization of the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system.

Sf9 cells infected with the recombinant mouse CaMKII-alpha (Ca2+/calmodulin dependent kinase II) baculovirus expressed 12-15 mg of MCaMKII-alpha per liter of cells. Approximately 50% of the MCaMKII-alpha activity could be purified using a CaM-Sepharose affinity column. The purified MCaMKII-alpha had a M(rapp) of 50 kDa by SDS-PAGE and a native Mr of 600 kDa. MCaMKII-alpha, like rat brain CaMKII, had an A0.5 for CaM of 100 nM, a Km for syntide-2 of 8 microM, and was able to generate Ca2(+)-independent activity by autophosphorylation. The baculovirus system expressed large quantities of MCaMKII-alpha with characteristics similar to the rat brain CaMKII, thus providing an expression system for the detailed biochemical analysis of MCaMKII-alpha.

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit.

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.

Regulation of brain Ca2+/calmodulin-dependent protein kinase II.

CaM-kinase II is a multifunctional protein kinase highly enriched in neural tissues where it modulates a variety of Ca2(+)-dependent processes. A complex regulatory domain in the kinase within residues 281-309 contains an autoinhibitory sequence, a CaM-binding region, and sites of regulatory autophosphorylation. Autophosphorylation on Thr286 converts the kinase to a Ca2(+)-independent form which could prolong physiological systems controlled by this kinase in response to transient Ca2+ elevations. Such properties appear to exist in dynamic equilibrium in the isolated postsynaptic density and in cultured brain cells. These unique biochemical regulatory properties, coupled with an unusual high concentration in the postsynaptic density of excitatory synapses, makes CaM-kinase II an attractive candidate for involvement in synaptic plasticity.

Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate.

A cDNA clone for the alpha subunit of mouse brain Ca2+/CaM-dependent protein kinase II (CaM-kinase II) was transcribed in vitro and translated in a rabbit reticulocyte lysate system. Inclusion of [35S]methionine in the translation system yielded a single 35S-polypeptide of about 50 kDa. When the translation system was assayed for CaM-kinase II activity, there was a 5-10-fold enrichment of kinase activity which was totally dependent on Ca2+/calmodulin (CaM). Both the 50-kDa 35S-polypeptide and the Ca2+/CaM-dependent protein kinase activity were quantitatively immunoprecipitated by rat brain CaM-kinase II antibody. When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca2+, CaM, Mg2+, and ATP, the Ca2+-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 +/- 0.7 to 26.5 +/- 2.1% of total activity (assayed in the presence of Ca2+/CaM). These properties confirm the identity of the kinase translated in vitro as CaM-kinase II. The role of Thr-286 autophosphorylation in formation of the Ca2+-independent activity was investigated by site-directed mutation of Thr-286 to Ala (Ala-286 kinase) and to Asp (Asp-286 kinase). The Ala-286 kinase was completely dependent on Ca2+/CaM for activity prior and subsequent to autophosphorylation. The Asp-286 kinase exhibited 21.9 +/- 0.8% Ca2+-independent activity, and this was not increased by autophosphorylation. These results establish that introduction of negative charge(s) at residue 286, either by autophosphorylation of Thr or by mutation to Asp, is sufficient and necessary to generate the partially Ca2+-independent form of CaM-kinase II.

Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation.

Regulatory mechanisms of rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) were probed using a synthetic peptide (CaMK-(281-309] corresponding to residues 281-309 (alpha-subunit) which contained the calmodulin (CaM)-binding and inhibitory domains and also the initial autophosphorylation site (Thr286). Kinetic analyses indicated that inhibition of a completely Ca2+/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive with respect to ATP. Interaction of CaMK-(281-309) with the ATP-binding site was independently confirmed since inactivation of proteolyzed CaM-kinase II by phenylglyoxal (t1/2 = 7 min) was blocked by ATP analog plus Mg2+ or by CaMK-(281-309). In the presence of Ca2+/CaM, CaMK-(281-309) no longer protected against phenylglyoxal inactivation, consistent with our previous observations (Colbran, R.J., Fong, Y.-L., Schworer, C.M., and Soderling, T.R. (1988) J. Biol. Chem. 263, 18145-18151) that binding of Ca2+/CaM to CaMK-(281-309) 1) blocks its inhibitory property, and 2) enhances its phosphorylation at Thr 286. The present study also showed that phosphorylation of CaMK-(281-309) decreased its inhibitory potency at least 10-fold without affecting its Ca2+/CaM-binding ability. Thus, CaM-kinase II is inactive in the absence of Ca2+/CaM because an inhibitory domain within residues 281-309 interacts with the catalytic domain and blocks ATP binding. Autophosphorylation of Thr286 results in a Ca2+/CaM-independent form of the kinase by disrupting the inhibitory interaction with the catalytic domain.

Regulatory interactions of the calmodulin-binding, inhibitory, and autophosphorylation domains of Ca2+/calmodulin-dependent protein kinase II.

Two peptide analogs of Ca2+/calmodulin-dependent protein kinase II (CaMK-(peptides)) were synthesized and used to probe interactions of the various regulatory domains of the kinase. CaMK-(281-289) contained only Thr286, the major Ca2+-dependent autophosphorylation site of the kinase (Schworer, C. M., Colbran, R. J., Keefer, J. R. & Soderling, T. R. (1988) J. Biol. Chem. 263, 13486-13489), whereas CaMK-(281-309) contained Thr286 together with the previously identified calmodulin binding and inhibitory domains (Payne, M. E., Fong, Y.-L., Ono, T., Colbran, R. J., Kemp, B. E., Soderling, T. R. & Means, A. R. (1988) J. Biol. Chem. 263, 7190-7195). CaMK-(281-309), but not CaMK-(281-289), bound calmodulin and was a potent inhibitor (IC50 = 0.88 +/- 0.7 microM using 20 microM syntide-2) of exogenous substrate (syntide-2 or glycogen synthase) phosphorylation by a completely Ca2+/calmodulin-independent form of the kinase generated by limited proteolysis with chymotrypsin. This inhibition was completely relieved by the inclusion of Ca2+/calmodulin in excess of CaMK-(281-309) in the assays. CaMK-(281-289) was a good substrate (Km = 11 microM; Vmax = 3.15 mumol/min/mg) for the proteolyzed kinase whereas phosphorylation of CaMK-(281-309) showed nonlinear Michaelis-Menton kinetics, with maximal phosphorylation (0.1 mumol/min/mg) at 20 microM and decreased phosphorylation at higher concentrations. The addition of Ca2+/calmodulin to assays stimulated the phosphorylation of CaMK-(281-309) by the proteolyzed kinase approximately 10-fold but did not affect the phosphorylation of CaMK-(281-289). A model for the regulation of Ca2+/calmodulin-dependent protein kinase II is proposed based on the above observations and results from other laboratories.

Dephosphorylation of the beta 2-adrenergic receptor and rhodopsin by latent phosphatase 2.

Recent evidence suggests that the function of receptors coupled to guanine nucleotide regulatory proteins may be controlled by highly specific protein kinases, e.g. rhodopsin kinase and the beta-adrenergic receptor kinase. In order to investigate the nature of the phosphatases which might be involved in controlling the state of receptor phosphorylation we studied the ability of four highly purified well characterized protein phosphatases to dephosphorylate preparations of rhodopsin or beta 2-adrenergic receptor which had been highly phosphorylated by beta-adrenergic receptor kinase. These included: type 1 phosphatase, calcineurin phosphatase, type 2A phosphatase, and the high molecular weight latent phosphatase 2. Under conditions in which all the phosphatases could dephosphorylate such common substrates as [32P]phosphorylase a and [32P]myelin basic protein at similar rates only the latent phosphatase 2 was active on the phosphorylated receptors. Moreover, a latent phosphatase activity was found predominantly in a sequestered membrane fraction of frog erythrocytes. This parallels the distribution of a beta-adrenergic receptor phosphatase activity recently described in these cells (Sibley, D. R., Strasser, R. H., Benovic, J. L., Daniel, K., and Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9408-9412). These data suggest a potential role for the latent phosphatase 2 as a specific receptor phosphatase.

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains.

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.

Identification and characterization of a phosphorylation-activated, cyclic AMP and Ca2+-independent protein kinase in the brain.

A cyclic AMP and calcium-independent protein kinase has been identified and purified from pig brain to near homogeneity. This independent protein kinase was isolated in an inactive form, and activation required ATP and Mg2+. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme contains 1 subunit with a molecular mass of about 36 kDa. Although there was no significant phosphorylation of phosphorylase, phosphorylase b kinase, casein, phosvitin, and protamine, this kinase was found to be very active toward myelin basic protein and histones H1, 2A, and 2B. Trypsinolysis completely destroyed the kinase activity, indicating that this is not a protease-activated protein kinase. More interesting, this cAMP and calcium-independent protein kinase can be regulated by its state of phosphorylation. In its non-phosphorylated state, the kinase was essentially inactive but could be fully activated when the enzyme was phosphorylated up to a 1:1 molar ratio. Conversely, partial dephosphorylation of the phosphorylated enzyme was associated with a time-dependent decrease in the kinase activity and a loss of 32P. All the results taken together point out that this kinase is distinguished from all the reported protein kinases and may represent a previously undiscovered protein kinase. The results also provide initial evidence that a cascade activation mechanism may possibly be involved in the regulation of a protein kinase activity which is independent of cAMP and calcium.

Endogenous basic protein phosphatases in the brain myelin.

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.

Purification and characterization of two inactive/latent protein phosphatases from pig brain.

Two inactive/latent protein phosphatases termed LP-1 (Mr 260,000) and LP-2 (Mr 350,000) were identified and purified from pig brain. Examination of molecular structures indicated that LP-1 has three subunits with molecular weights of 69,000, 55,000, and 34,000, respectively, whereas LP-2 contains only one subunit, with molecular weight of 49,000. When using phosphorylase a as a substrate, LP-1 was completely inactive and could be dramatically activated by freezing and thawing in 0.2 M 2-mercaptoethanol, whereas LP-2 contained some basal activity but could also be stimulated 40-fold by the same treatment. Kinetic analysis further indicated that both LP-1 and LP-2 enzymes dephosphorylate histone 2A, myelin basic protein, and phosphorylase a at a rather comparable rate, but the dephosphorylation of histone 2A and myelin basic protein seems to be spontaneously active. This, together with the results that trypsinolysis could specifically knock off phosphorylase phosphatase activity but caused no effect on the associated myelin basic protein/histone phosphatase activities, supports the notion that a two-site mechanism may possibly be involved in the regulation of substrate specificity of LP-1 and LP-2 enzymes in the central nervous system.

Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain.

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32P-labeled myelin basic protein (MBP) and [32P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues.