Purification of a Fatty Acid-stimulated Protein-serine/threonine Phosphatase from Bovine Brain and Its Identification as a Homolog of Protein Phosphatase 5*

An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with aM r of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 μm phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4–14-fold, with half-maximal stimulation at 50–100 μm, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator.

subunits of PP1, PP2A, and calcineurin constitute a large family of structurally related enzymes (1)(2)(3). Cloning studies have also revealed several novel protein-Ser/Thr phosphatases related to PP1 and PP2A, the properties and functions of which are not yet understood (2). In addition to a catalytic subunit, PP1, PP2A, and calcineurin each contain one or more regulatory subunits. Mechanisms regulating the activity of these enzymes include the control of localization and substrate specificity by regulatory subunits; direct phosphorylation; the binding of a second messenger; and inhibition by regulatory domains, subunits, and interacting proteins (2). Several members of the PP1/PP2A family have been shown to be sensitive to putative lipid second messengers (4 -8).
Arachidonic acid and several of its lipoxygenase or cytochrome P-450 metabolites have been reported to modulate the activity of a variety of ion channels (9 -13). Although ion channel responses may be directly regulated by lipids (9,10), in several cases, lipid-dependent responses can be blocked by Ser/Thr phosphatase inhibitors such as microcystin, okadaic acid, and calyculin (11)(12)(13). This suggests that a Ser/Thr phosphatase related to PP1 or PP2A may mediate the effect of some lipids on ion channel activity (14). The possibility that a lipidactivated Ser/Thr phosphatase may in turn regulate ion channel function prompted us to look for such an enzyme. In this report, we describe the purification of a fatty acid-stimulated Ser/Thr phosphatase from bovine brain. The purified enzyme contains a single M r 63,000 polypeptide and is identical or closely related to the recently cloned Ser/Thr phosphatase PP5 (15)(16)(17). 2 We also demonstrate that recombinant rat PP5 is similarly activated by the unsaturated fatty acid arachidonic acid (AA). Protein phosphatase 5 and its yeast homolog (PPT1) contain a C-terminal catalytic domain that is structurally related to PP2A and PP1 and an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) that is not shared with other members of the PP1/PP2A family (15)(16)(17). The stimulation of PP5 in vitro by unsaturated fatty acids suggests that this enzyme may be regulated in vivo and raises the possibility that it is a target for activation by a lipid second messenger or some other endogenous effector. * This work was supported in part by National Institutes of Health Grants NS31221 (to S. R.) and CA59935 (to H. C.), American Heart Association Grant 3483670 (to S. R.), and an American Cancer Society institutional grant (to the Purdue Cancer Center). Work performed in the Purdue Laboratory for Macromolecular Structure (peptide sequencing and oligonucleotide synthesizing) was supported in part by National Institutes of Health Grants DK20542 and CA23168. Work performed in the DNA Sequencing Facility of the Purdue Cancer Center (cDNA sequencing) was supported by National Institutes of Health Grant CA23168. This is Journal Paper 15315 from the Purdue University Agricultural Experimental Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2 Protein phosphatase 5 cloned from a human teratocarcinoma cell cDNA library (16), PPT cloned from a rat fat cell cDNA library (15), and PPK cloned from a mouse lung cDNA library (17) are Ͼ90% identical in amino acid sequence, but PP5 is only 40% identical to PPT1 cloned from S. cerevisiae (16). We will therefore refer to mammalian forms of this enzyme as PP5 and reserve the term PPT for the yeast form of the enzyme. This nomenclature is consistent with the majority of other reports on this enzyme (18 -23) following the initial cloning studies.

EXPERIMENTAL PROCEDURES
Materials and Purified Proteins-All materials were purchased from Sigma unless otherwise noted. The catalytic subunit of cAMP-dependent protein kinase was purified from bovine heart (24). Protein phosphatase 2A containing a M r 55,000 BЈ subunit (25,26) was purified from rat brain. 3 Purification of a M r 63,000 Protein-Ser/Thr Phosphatase-All steps were carried out at 4°C unless otherwise noted. Frozen bovine brain cortex (615 g) was thawed and homogenized in a Waring blender with 1.2 liters of 20 mM Tris-HCl, pH 7.6 (4°C), 1 mM EDTA, 1 mM EGTA, 0.1% ␤-mercaptoethanol, 0.5 g/ml leupeptin, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, and 1 M pepstatin A (buffer A). The homogenization buffer also included 10 g/ml trypsin inhibitor. The homogenate was centrifuged at 15,000 ϫ g for 40 min, and the supernatant was filtered through glass wool, diluted to 3 liters, and then mixed with 500 ml of DE52 (Whatman) for 90 min. The DE52 was washed until the effluent absorbance at 280 nm was equal to that of the wash buffer, and then bound proteins were eluted with 1 liter of buffer A containing 200 mM NaCl and 10 g/ml trypsin inhibitor. Eluted protein was dialyzed against buffer A and then applied to a 5 ϫ 30-cm DEAE-Sepharose CL-6B column. The column was eluted with a 1-liter gradient of 0 -0.3 M NaCl in buffer A (including 10 g/ml trypsin inhibitor) at a flow rate of 7.5 ml/min. Fractions of 7.5 ml were collected and assayed as described below. A peak of AA-stimulated phosphatase activity eluting at 60 mM NaCl was pooled, dialyzed in buffer A, and then applied to a 2.5 ϫ 18-cm CM-Sepharose CL-6B column and eluted with a 300-ml gradient of 0 -0.3 M NaCl in buffer A at a flow rate of 2 ml/min. Fractions of 2.5 ml were collected and assayed. A single peak of phosphatase activity eluting at 100 mM NaCl was pooled and diluted with buffer A to contain ϳ25 mM NaCl and then loaded onto a 1 ϫ 14-cm heparin-agarose column. The column was washed with 15 ml of buffer A containing 100 mM NaCl and then eluted with a 10-ml gradient of 0.1-1 M NaCl in buffer A. Fractions of 0.5 ml were collected at 0.5 ml/min and assayed. Active fractions were pooled and subjected to gel filtration on a 1.5 ϫ 95-cm Sephadex G-100 superfine column (Pharmacia Biotech Inc.) in buffer A containing 100 mM NaCl. Standards used to calibrate the column included aldolase (158 kDa), bovine serum albumin (66.2 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), and aprotinin (6.5 kDa). The column flow rate was 0.1 ml/min, and 1-ml fractions were collected and assayed. Active fractions were pooled and loaded onto a 1.5 ϫ 16-cm ␣-casein-agarose column. This column was washed and then eluted with a 100-ml gradient of 0 -0.3 M NaCl in buffer A at 1 ml/min; fractions of 1 ml were collected and assayed. A single peak of AA-stimulated phosphatase activity eluting at 140 mM NaCl was pooled and concentrated on a 1-ml heparin-agarose column and then subjected to cation-exchange chromatography on a Mono S HR 5/5 column (Pharmacia Biotech Inc.) at 25°C. The column was washed and eluted with a 110-ml gradient of 0 -186 mM NaCl in buffer A (pH 7.6, 25°C) at a flow rate of 1 ml/min. Fractions of 1 ml were collected at 4°C and assayed for phosphatase activity. Pooled fractions were dialyzed against buffer A containing 50% glycerol and stored at Ϫ20°C. Purification was monitored by SDS-polyacrylamide gel electrophoresis (27) using a 4% stacking gel and a 10% resolving gel, and proteins were detected by staining with Coomassie Blue or silver (28).
Preparation of 32 P-Labeled Substrates-Casein, RCML (Life Technologies, Inc.), and lysine-rich histone (type III-S from calf thymus, Sigma) were phosphorylated overnight at 30°C in a reaction containing 20 mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 0.1% ␤-mercaptoethanol, 750 M [␥-32 P]ATP (3.5 ϫ 10 14 cpm/mol ATP), 5 mg/ml substrate, and 4 g/ml purified catalytic subunit of cAMP-dependent protein kinase (29). Each reaction was terminated by adding trichloroacetic acid to a final concentration of 12% (w/v) and then left on ice for 2 h and centrifuged at 12,000 ϫ g for 10 min at 4°C. The resulting pellet was resuspended in 1 M Tris-HCl, pH 7.6, and dialyzed against 50 mM Tris-HCl, pH 7.6, and 0.1 mM EGTA at 4°C. Myelin basic protein (Life Technologies, Inc.) was phosphorylated as described above in a 500-l reaction, and then the reaction was terminated with 800 l of ice-cold 20% (w/v) trichloroacetic acid in 20 mM NaH 2 PO 4 . The mixture was left for 15 min on ice and centrifuged for 15 min at 4°C in a microcentrifuge. The pellet was washed four times; resuspended in 1 M Tris-HCl, pH 7.6 (4°C); and dialyzed as described above. Kemptide was phosphorylated in the same manner as MBP and purified by binding to phosphocellulose P-81 paper (29). Phosphorylase a was phosphorylated as described by Cohen et al. (30). The average stoichiometry of phos-phorylation for these substrates (expressed as mol of phosphate/mol of protein) was 0.27 for casein, 0.21 for RCML, 0.2 for histone, 0.96 for MBP, 0.33 for Kemptide, and 0.4 for phosphorylase a. During purification, casein was phosphorylated with 100 M [␥-32 P]ATP (3.5 ϫ 10 15 cpm/mol ATP) to a stoichiometry of 0.05 mol of phosphate/mol of casein and used to assay column fractions and pools.
Phosphatase Assays-Phosphatase reactions conducted during purification were carried out for 15 min at 30°C with 0.6 M 32 P-casein in the presence and absence of 50 M AA (Calbiochem) in 60 mM Tris, pH 7.6 (25°C), 1 mM EDTA, 1 mM EGTA, and 0.1% ␤-mercaptoethanol (buffer B). Reactions were initiated by adding 20 l of substrate containing lipid or ethanol vehicle to 10 l of an enzyme sample. Once the phosphatase was purified, the assay conditions were modified slightly. All solutions were pre-equilibrated to room temperature just before assay. Ten microliters of lipid or ethanol vehicle solution were mixed with 10 l of phosphatase, and then the reaction was immediately initiated by adding 10 l of substrate and incubated for 10 min at 30°C in buffer B. Reactions were terminated with 100 l of ice-cold 10% (w/v) trichloroacetic acid and 20 l of 7.5 mg/ml bovine serum albumin and then microcentrifuged at 12,000 ϫ g for 5 min. Reactions with 32 P-RCML were terminated instead with 100 l of ice-cold 20% (w/v) trichloroacetic acid. Assays using 32 P-Kemptide, 32 P-histone, and 32 P-MBP were quenched with 450 l of 4% (w/v) activated charcoal in 0.6 M HCl, 90 mM Na 4 P 2 O 7 , and 2 mM NaH 2 PO 4 and then centrifuged at 12,000 ϫ g for 5 min. Acid-soluble 32 P i was quantified by liquid scintillation counting. The phosphate released from each substrate was Ͻ20% of the total present. The final ethanol concentration in lipid-containing reactions was 0.8 -1.7% and had no significant effect on phosphatase activity.
Fluorometric Determination of the Lipid Critical Micellar Concentration-The CMCs of lipid solutions in 60 mM Tris, pH 7.6 (25°C), 1 mM EDTA, 1 mM EGTA, 0.1% ␤-mercaptoethanol, 1.3% ethanol, and a 2 M concentration of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene were determined by fluorescence spectroscopy (31). Lipids were dispersed by sonication or vigorous vortexing, equilibrated to room temperature, and then incubated with fluorescent probe in the dark for at least 30 min. Fluorescence intensity was measured at excitation and emission wavelengths of 358 and 430 nm, respectively, on a Hitachi F-2000 fluorescence spectrophotometer.
Peptide Sequencing and Analysis-Fifty micrograms of purified phosphatase were concentrated by trichloroacetic acid precipitation, resuspended in 8 M urea and 0.4 M NH 4 HCO 3 , reduced and carboxymethylated (32), and then digested with 1.8 g of endoproteinase Lys-C (Waco Chemicals) at 37°C overnight. Peptides were purified by reversephase HPLC on a Vydac C 18 column (2.1 ϫ 250 mm) using a gradient of 0 -30% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 150 l/min. The amino acid sequences of selected peptides were determined by automated Edman degradation performed with either an Applied Biosystems 470A or 491 gas-phase sequencer. A BLAST search (33) was performed to compare the peptide sequences obtained with those in the GenBank™, SwissProt, and PIR data bases.
Cloning and Expression of PP5-Messenger RNA was isolated from rat GH 4 C 1 cells and used to generate cDNA. The cDNA was then used as a template in polymerase chain reaction together with PP5-specific oligonucleotide primers, and the major polymerase chain reaction product was cloned into the MluI-NotI sites of the mammalian expression vector pCI (Promega). The 1.5-kilobase pair cDNA containing the entire PP5 coding region was then amplified by polymerase chain reaction using the pCI-PP5 clone as a template. The 5Ј-sense primer (5Ј-GACT-GGATCCATGGCGATGGCGGAGGGCGAG) and the 3Ј-antisense primer (5Ј-GACTGAATTCTTACATCATTCCTAGCTG) utilized in this polymerase chain reaction contained initiation and stop codons, respectively, as well as BamHI and EcoRI restriction linkers. The major product containing the full-length PP5 coding region was then cloned into the BamHI-EcoRI sites of pBluescript II (pBII) KS Ϫ (Stratagene) and sequenced using an ALFexpress automatic DNA sequencer (Pharmacia Biotech Inc.) to verify its authenticity. To perform bacterial expression, the entire PP5 cDNA was excised from the pBII-PP5 construct using BamHI and EcoRI restriction enzymes and cloned immediately downstream of the GST coding region into the BamHI-EcoRI sites of the pET-21a GST plasmid. 4 The newly constructed plasmid, termed pET GST-PP5, was then transformed into Esherichia coli strain BL21(DE3). Bacterial cultures were grown in Luria broth supplemented with 50 g/ml ampicillin and 4 mM MnCl 2 , and protein expres-sion was induced with 50 M isopropylthiogalactoside. Cells were lysed in 50 mM Tris, pH 7.6, 0.1% ␤-mercaptoethanol, 2 mM EDTA, 4 mM MnCl 2 , 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml each aprotinin, leupeptin, and pepstatin. The GST-PP5 fusion protein was affinitypurified with glutathione-agarose and then treated with thrombin (34,35). Proteolysis was terminated with 1 mM phenylmethylsulfonyl fluoride, and recombinant PP5 was dialyzed in 50% glycerol, 1 mM EGTA, 0.1% ␤-mercaptoethanol, 20 mM Tris, pH 7.6 (4°C), and 4 mM MnCl 2 overnight and then stored at Ϫ20°C. The amino acid sequence of recombinant PP5 is identical to that reported by Becker et al. (15) except for four residues (GSGS) remaining at the N terminus after thrombin cleavage.
Determination of Catalytic Parameters with pNPP-Recombinant PP5 was assayed for para-nitrophenylphosphatase activity in a 50-l reaction containing 10 -150 mM pNPP, 500 ng of enzyme, and 0 -40 M AA in 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1% ␤-mercaptoethanol, and 0.1% ethanol. Aliquots of enzyme were preincubated for 1 min at 30°C, and then reactions were initiated by adding a mixture of substrate and lipid prewarmed to 30°C. Control reactions were assayed without enzyme to determine the spontaneous hydrolysis of pNPP at each substrate concentration. Assays were terminated after 15 min with 450 l of 0.25 N NaOH. Sample absorbance was measured at A 410 to quantitate the release of para-nitrophenolate, after subtracting the appropriate background (29). The catalytic parameters K m and V max were determined by nonlinear regression and Lineweaver-Burk analysis using the computer software program EnzymeKinetics (Trinity Software).

RESULTS
Detection and Purification of a Fatty Acid-stimulated Ser/ Thr Phosphatase from Soluble Brain Extract-When soluble extracts prepared from rat brain were subjected to Mono Q anion-exchange chromatography and fractions were assayed for phosphatase activity using 32 P-casein as substrate in the presence of 50 M AA, a peak of activity eluting at ϳ110 -125 mM NaCl was observed (data not shown). Its activity ranged from undetectable to very low in the absence of AA. A similar peak of lipid-stimulated phosphatase activity was observed during chromatography of soluble extracts from bovine brain or heart and from rat clonal GH 4 C 1 pituitary cells. After Mono Q ion-exchange chromatography, the lipid-stimulated activity was inhibited Ͼ80% by 100 nM okadaic acid, suggesting that it was related to PP1 or PP2A (36).
To purify the fatty acid-stimulated phosphatase, a soluble extract was prepared from bovine brain and subjected to batch chromatography on DE52-cellulose, followed by column chromatography on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, heparin-agarose, Sephadex G-100 superfine, ␣-casein-agarose, and Mono S (Table I). Phosphatase activity was monitored in the presence and absence of 50 M AA using 32 P-casein as a substrate. A single peak of AA-stimulated phosphatase activity eluting at 60 mM NaCl was resolved from the majority of phosphatase activity during chromatography on DEAE-Sepharose CL-6B. During gel filtration, the lipid-stimulated phosphatase eluted with an apparent M r of 63,000. After Mono S chromatography, the purified phosphatase contained a single M r 63,000 band when analyzed by SDS-polyacrylamide gel electrophoresis and staining with silver ( Fig. 1) or Coomassie Blue (data not shown). These results suggested that the lipidstimulated phosphatase is a monomer of M r 63,000 and that it is distinct from PP1 and PP2A, which are typically multisubunit enzymes containing catalytic subunits of 36 -37 kDa (1-3). Four-hundred micrograms of pure phosphatase were obtained from 615 g of bovine brain (Table I). The pure enzyme exhibited a specific activity of 6 nmol of P i released per min/mg of enzyme when assayed in the presence of 50 M AA and 0.6 M 32 Pcasein for 15 min at 30°C (Table I).
Identification of the Fatty Acid-stimulated Phosphatase as a Homolog of PP5-The purified phosphatase was inhibited by okadaic acid (IC 50 ϭ 6 nM) and microcystin (IC 50 ϭ 4 nM). The sensitivity of the lipid-stimulated enzyme to these inhibitors (36), together with its apparent molecular weight, suggested that it may represent a recently cloned protein-Ser/Thr phosphatase, PP5 (15)(16)(17). Protein phosphatase 5 has a predicted molecular mass of 58 kDa, and recombinant human PP5 is inhibited by okadaic acid (IC 50 ϭ 1.5 nM) and microcystin (IC 50 ϭ 1 nM) (16) when assayed at an enzyme concentration similar to that used in the present study.
To determine its identity, the amino acid sequences of peptides derived from the lipid-stimulated phosphatase were obtained. The purified enzyme was digested with endoproteinase Lys-C, and then proteolytic peptides were purified by HPLC. Five peptides were sequenced and found to correspond to regions of PP5. An alignment of these peptides with corresponding sequences deduced from the rat PP5 cDNA (15) is shown in Fig. 2. Four of the peptides (peptides B-E in Fig. 2) correspond to regions within the catalytic domain of PP5, which is itself  homologous to the catalytic subunits of PP1, PP2A, and calcineurin (15)(16)(17). These peptides are 96% (B), 89% (C), 100% (D), and 84% (E) identical to the corresponding residues of rat PP5. The remaining peptide (peptide A in Fig. 2) is similar to a stretch of sequence in the N-terminal TPR domain of PP5. This peptide is 91% identical to the corresponding sequence deduced from the rat PP5 cDNA. Since the TPR domain exists in PP5, but not in other known Ser/Thr phosphatases (15)(16)(17), the identification of peptide A establishes that the lipid-stimulated phosphatase is most closely related to PP5, rather than other members of the PP1/PP2A family. Human PP5 (16) and its homologs cloned from a mouse lung cDNA library (17) and a rat fat cell cDNA library (15) are completely conserved in the residues corresponding to peptides A-E. We will refer to the enzyme purified from bovine brain as a PP5 homolog in this report. Effect of AA on Dephosphorylation of Casein and Other Substrates-Stimulation of purified phosphatase activity by AA was concentration-dependent. When assayed with 1 or 2.5 M 32 P-casein, half-maximal stimulation occurred at ϳ50 M AA, with 6-fold maximal stimulation (Fig. 3). At these low substrate concentrations, the response of the enzyme to lipid was biphasic, with stimulation declining above 250 M lipid. At higher levels of substrate (5-10 M), the concentration of lipid required for half-maximal stimulation was increased 2-fold, greater maximal activation (9 -14-fold) was observed, and the biphasic nature of the stimulatory effect was diminished. The loss of this biphasic effect and the increase in lipid concentration required for half-maximal activation may be due to the binding of lipid by casein.
The activity of the PP5 homolog toward other substrates was also stimulated by AA. In the absence of lipid, phosphatase activity toward histone, MBP, and RCML was comparable to that toward casein, ranging from 24 to 62 nmol of P i released per min/mg of phosphatase when assayed with 10 M phosphosubstrate (Table II). Arachidonic acid stimulated phosphatase activity toward all of these substrates in a concentration-dependent manner, although its effect was less pronounced than on activity for casein. Half-maximal stimulation by lipid occurred between 75 and 100 M lipid, and maximal stimulation ranged from 4 to 12-fold, with dephosphorylation of histone being the least sensitive and dephosphorylation of MBP the most sensitive. As with casein, a biphasic response to lipid was observed for MBP; however, stimulation of histone or RCML dephosphorylation reached a plateau at high lipid concentrations. Since AA can increase the activity of the bovine brain PP5 homolog for a variety of substrates, the stimulatory effect of lipid is likely to be enzyme-directed. Little or no activity was seen with phosphorylase a or Kemptide.
Structural Requirements for Lipid Stimulation-To determine whether the activating lipid demonstrates specific structural requirements, the ability of various fatty acids or fatty acid analogs to stimulate the activity of the purified phosphatase toward 32 P-casein or 32 P-RCML was assessed. At 200 M, the unsaturated fatty acids AA, oleic acid, linoleic acid, and palmitoleic acid each stimulated phosphatase activity 4 -9-fold toward 32 P-casein and 4 -6-fold toward 32 P-RCML (Table III); AA and oleic acid were the most potent. Methyl ester or alcohol derivatives of AA and oleic acid had little or no effect on phosphatase activity. The saturated fatty acids arachidic acid, stearic acid, and caproic acid did not significantly affect activity at concentrations of 200 M, whereas myristic acid and lauric acid slightly stimulated activity toward 32 P-RCML. These results suggest that stimulation by fatty acids is not simply a hydrophobic effect and that the carboxyl moiety and at least FIG. 2. Sequence analysis. A, the sequences of five proteolytic fragments generated from the purified bovine brain lipid-stimulated phosphatase are compared with corresponding residues from the deduced sequence of rat PP5 (as reported by Becker et al. (15), GenBank™/EMBL Data Bank accession number X77237). Cycles in which the major peak corresponded to the approximate migration of carboxymethylated cysteine are denoted c. B, presented is a diagrammatic representation of the catalytic and TPR domains of PP5 showing the location of sequences corresponding to each peptide in A.
FIG. 3. Concentration-dependent stimulation of the bovine brain PP5 homolog by AA. Phosphatase activity was assayed for 10 min at 30°C as described under "Experimental Procedures." Reactions included 1 (q), 2.5 (E), 5 (ϫ), or 10 (f) M 32 P-casein, 4 nM purified phosphatase, and varying concentrations of AA. Data are presented as the average nmol of P i released per min/mg of phosphatase Ϯ S.E. Values at each substrate concentration are from three independent assays performed in triplicate. one double bond are required for stimulation.
The concentration of AA at which half-maximal activation occurred corresponds to its CMC, 50 M, measured under conditions similar to those used in the phosphatase assay. Other activating fatty acids exhibited similar CMCs under these conditions: oleic acid, 43 M; linoleic acid, 92 M; and palmitoleic acid, 70 M. In contrast, arachidic acid and the methyl ester of oleic acid did not significantly stimulate activity at 200 M (Table III), which is above their CMCs measured under the assay conditions used in this study. This suggests that unsaturated fatty acids in micellar form activate the PP5 homolog, but that micelle formation alone is not sufficient.
Recombinant PP5 Is Activated by AA-To determine whether PP5 is activated by AA, a cDNA encoding rat PP5 was expressed in bacteria as a cleavable GST fusion protein, which was then affinity-purified, cleaved from carrier GST, and tested for sensitivity to lipid. The nucleotide sequence of the rat PP5 cDNA generated from GH 4 C 1 cells is identical to that of the PP5 cDNA isolated from a rat fat cell cDNA library (15) that encodes a protein of 58 kDa. After affinity purification with glutathione-agarose and subsequent thrombin cleavage, the recombinant enzyme migrated as a single polypeptide of M r 63,000 on Coomassie Blue-stained SDS-polyacrylamide gels (data not shown). On SDS gels, the purified bovine brain PP5 homolog and recombinant rat PP5 have nearly identical mobilities. Recombinant rat PP5 was also similar to the bovine brain PP5 homolog in its sensitivity to inhibition by okadaic acid or microcystin (data not shown). While the concentration of AA required for half-maximal stimulation was the same for recombinant PP5 and the purified bovine brain PP5 homolog, maximal stimulation of recombinant PP5 ranged from 20 to 25-fold, compared with ϳ14-fold maximal stimulation seen with the purified enzyme (Fig. 4A). This difference may occur due to species variation in structure, or a decreased responsiveness may result from the more complicated and prolonged purification required in the case of the bovine brain PP5 homolog. As with the purified bovine brain PP5 homolog, stimulation of recombinant PP5 by AA was observed with both 32 P-casein and FIG. 4. Stimulation of recombinant PP5 or the bovine brain PP5 homolog by AA. A, the phosphatase activity of the purified PP5 homolog (E) or recombinant rat PP5 (q) was assayed for 10 min at 30°C with 10 M 32 P-casein and increasing concentrations of AA as described under "Experimental Procedures." Data are presented as the activity relative to control in the absence of added lipid. Values are the average Ϯ S.D. from a single assay performed in triplicate. The experiment was performed twice with similar results. B, phosphatase activity was assayed for 10 min at 30°C with 10 M 32 P-casein or 32   Phosphatase activity was assayed for 10 min at 30°C with 4 nM purified phosphatase, 250 M arachidonic acid or vehicle, and 10 M 32 P-labeled substrate as described under "Experimental Procedures." Lipid was diluted from a 60 mM stock in 100% ethanol into an argonpurged buffer containing 20 mM Tris-HCl, pH 7.6, and vortexed vigorously under argon just before assays. Data are presented as the average nmol of P i released per min/mg of enzyme Ϯ S.E., and the activity of arachidonic acid-treated samples is presented relative to the corresponding vehicle control. Values represent three independent assays performed in triplicate.

TABLE III Effects of fatty acids and analogs on purified phosphatase activity
Phosphatase activity was assayed with 10 M 32 P-RCML or 32 Pcasein, 4 nM purified phosphatase (7.5 ng/reaction), and 200 M lipid or vehicle as described under "Experimental Procedures." Data are presented for the lipid-treated samples relative to the corresponding ethanol vehicle control. The values presented are the average Ϯ S.E. from at least three independent assays performed in triplicate. Control activity assayed with 32 P-RCML released 59.2 Ϯ 8.7 nmol of P i /min/mg of enzyme, whereas controls assayed with 32 P-casein released 49.2 Ϯ 5.9 nmol of P i /min/mg of enzyme.  32 P-MBP (Fig. 4B). Thus, recombinant PP5 is similar in its properties to the PP5 homolog purified from bovine brain. In contrast, under conditions in which AA activated PP5 14-fold with 32 P-casein, PP2A containing a M r 55,000 BЈ subunit was only modestly stimulated (Fig. 5). Stimulation of recombinant PP5 by AA was also observed in assays with the substrate pNPP. As with phosphoprotein substrates, stimulation of PP5 activity for pNPP was biphasic, although some differences were noted. Half-maximal stimulation by AA occurred at ϳ50 M, a 5-fold maximal stimulation was seen at 100 M, and higher lipid concentrations led to a sharper decline in stimulation (Fig. 6A). These results obtained with an aryl phosphomonoester are consistent with an effect of lipid that is enzyme-directed. Since pNPP could be assayed at a high molar ratio of substrate to enzyme and contained a single hydrolyzable phosphate group, this substrate was used to examine the effect of lipid on catalysis by PP5. Control PP5 activity for pNPP showed Michaelis-Menten kinetics, yielding a K m of 27 mM and a V max of 660 pmol of P i released per min (Fig. 6B). Arachidonic acid caused a modest decrease in K m and an increase in V max at lipid concentrations below the observed CMC. At 40 M AA, we observed a K m of 10 mM and a V max of 1400 pmol of P i released per min, leading to a 6-fold increase in catalytic efficiency (k cat /K m ) from 47 to 270 M Ϫ1 s Ϫ1 .
Effect of Salt on PP5 Activity-Control and lipid-stimulated PP5 activities were each affected by NaCl and KCl in a concentration-dependent manner. When the recombinant enzyme was assayed in the presence and absence of 150 mM KCl, control activity was inhibited ϳ50% by KCl, and the concentration of AA required for half-maximal stimulation was increased 1.7-fold in the presence of salt (data not shown). The threshold for stimulation by AA and the maximal activity with AA were not altered by salt, and the CMC for AA (50 M) was unaffected by salt (data not shown). Thus, at an ionic strength similar to intracellular conditions, AA stimulates PP5 as much as 40-fold over control values. DISCUSSION We have purified a M r 63,000 monomeric fatty acid-activated Ser/Thr phosphatase from soluble extracts of bovine brain and have identified this enzyme as a homolog of PP5. The bovine brain PP5 homolog is activated by AA over a 10-fold range in concentration, with half-maximal stimulation occurring at 50 -100 M. Stimulation by AA and other unsaturated fatty acids is observed with a variety of substrates, indicating that lipid activation represents a direct effect on the enzyme itself. The response of recombinant rat PP5 to AA is similar to that of the purified phosphatase from bovine brain, consistent with our conclusion that the bovine brain enzyme is similar or identical to PP5. Others have also shown that recombinant human PP5 and native PP5 partially purified from rabbit liver can be stimulated by polyunsaturated fatty acids (37). Little is known concerning how the activity of PP5 is controlled. The activation of PP5 by unsaturated fatty acids reveals this enzyme's potential for regulation and suggests that PP5 may be controlled in vivo by a bioactive lipid or by some other endogenous activator.
Activation by AA is not a general property of the PP1/PP2A family since PP2A which contains a M r 55,000 BЈ subunit is only modestly stimulated by AA. However, at least two other FIG. 5. Stimulation of the bovine brain PP5 homolog or PP2A by AA. The phosphatase activity of the purified PP5 homolog (E) or heterotrimeric PP2A containing a M r 55,000 BЈ subunit (q) was assayed for 10 min at 30°C with 10 M 32 P-casein and increasing concentrations of AA as described under "Experimental Procedures." Data are presented as the activity relative to control in the absence of added lipid. Values at each lipid concentration are the average Ϯ S.E. from three independent assays performed in triplicate. Control activity was 48 nmol of P i released per min/mg of enzyme for the PP5 homolog and 627 nmol of P i released per min/mg of enzyme for PP2A.
FIG. 6. Stimulation of recombinant PP5 activity for pNPP by AA. A, rat PP5 phosphatase activity was assayed for 30 min at 30°C with 250 ng of enzyme, 50 mM pNPP, and 0 -500 M AA as described under "Experimental Procedures." Results are shown as the average mol of P i released per min/mg of enzyme Ϯ S.E. from a representative assay performed in triplicate. The experiment was performed twice with similar results. Control PP5 activity released 920 Ϯ 6 nmol of P i /min/mg of enzyme. B, phosphatase activity was assayed for 15 min at 30°C with 500 ng of rat PP5, 10 -150 mM pNPP, and 0 -40 M AA as described under "Experimental Procedures." The Lineweaver-Burk double-reciprocal plot shows the average values for control (OE) and 10 (Ⅺ), 20 (f), 30 (E), or 40 (q) M AA-treated samples from three independent assays. The percentage of error for velocity averaged Ͻ3% and was independent of lipid and substrate concentration. Weighted (fourth power) least-squares fitting was used to estimate K m and V max . phosphatases in the PP1/PP2A family are sensitive to AA: smooth muscle myosin light chain phosphatase (SM-PP1M), which is a form of PP1 (4), and an unidentified form of PP2A. 5 SM-PP1M, which responds to a similar range of AA concentrations in vitro as that required for PP5 activation, is a potential physiologic target for AA in mediating the sensitizing effect of calcium on smooth muscle contraction (38). Treatment of SM-PP1M with AA decreases dephosphorylation of heavy meromyosin, but increases activity for another substrate, phosphorylase a (4). Physiologic targets for PP5 have not yet been defined. Although we have observed stimulation with a number of artificial substrates, it remains possible that AA or some other effector may inhibit, rather than stimulate, dephosphorylation of a physiologically relevant substrate by PP5. In the case of SM-PP1M, AA dissociates two regulatory subunits from the catalytic subunit, which itself is insensitive to AA (4). A region of the 130-kDa M subunit of SM-PP1M that may mediate the response to AA has recently been identified (39). A region corresponding to this stretch of sequence is not obviously present in PP5.
Protein phosphatase 5 and PPT1, its homolog from the yeast Saccharomyces cerevisiae, are distinguished from other members of the PP1/PP2A family in containing a unique N-terminal domain consisting of several TPRs (15)(16)(17). Tetratricopeptide repeats are hypothesized to form amphipathic helices (40) and have been shown to mediate protein-protein interactions (41). Although the locus of fatty acid binding is not known, it is tempting to speculate that the putative amphipathic helices of the N-terminal TPR domain may confer lipid sensitivity to PP5 since amphipathic helices have the potential to interact with lipid bilayers (42). Unsaturated fatty acids may disrupt interaction between the TPR domain and the catalytic domain of PP5 in a manner analogous to the dissociation of SM-PP1M subunits by AA (4). Alternatively, lipids may promote an activating interaction between these domains or may mimic the binding of an activating protein. Protein phosphatase 5 has been shown to bind an atrial natriuretic factor receptor in vitro (17) and to interact with glucocorticoid receptors and hsp90 in vivo (23); however, no effect of these interacting proteins on phosphatase activity was reported.
It remains to be established whether AA, other bioactive lipids, or some other biomolecule regulates PP5 in vivo. The levels of lipid required in this study are higher than those required to activate protein kinase C, a potential intracellular target for AA activation (43), but are similar to the levels of fatty acid shown to affect the activity of SM-PP1M (4), G z ␣ (44), Ras GTPase-activating protein (45), and Rac (46). These high levels may be required to mimic the action of a more potent lipid or a protein activator. Alternatively, in the context of a membrane or in combination with some other signal, the sensitivity of PP5 to AA may be enhanced. It will be important to evaluate the effects of other bioactive lipids on PP5 and to analyze the structural basis of lipid activation.
Arachidonic acid and its metabolites participate in cellular functions such as secretion (47,48), apoptosis (49), and mitogenesis (50). In addition, as mentioned earlier, a Ser/Thr phosphatase has been implicated in ion channel regulation by various AA metabolites. Protein phosphatase 5 may be a candidate for mediating the effects of AA or one of its metabolites in one or more of these lipid-sensitive processes. Since protein kinase C is a potential intracellular target for AA (43), AA, like calcium, may control two opposing processes, the phosphorylation and dephosphorylation of Ser/Thr residues.
Regulation of ion channels or hormone receptors would re-quire that PP5 be present in the cytoplasm or associated with the plasma membrane. Chen et al. (16) observed that PP5 is predominantly nuclear in normal and transformed fibroblasts. However, the association of PP5 with atrial natriuretic factor receptors (17) and complexes containing hsp90 and glucocorticoid receptors (23) suggests that, under some circumstances, PP5 may be localized to the cytoplasm or the plasma membrane. To understand the biological function of PP5, it will be important to determine whether PP5 is regulated by a lipid second messenger or some other effector under physiological circumstances, to establish whether PP5 is present in other subcellular compartments in addition to the nucleus, and to define its intracellular targets.