THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U.S.A. Vol. 256. No. 15, Issue of August 10, pp, 8197-8201, 1981

Selective Dephosphorylation of Proteins Containing Phosphotyrosine by Alkaline Phosphatases*

(Received for publication, January 28, 1981, and in revised form, April 6, 1981)

Ghanshyam SwarupS, Stanley Cohengv, and David L. GarbersSII ** From the Departments of +Pharmacology, I)Physiology, and IBiochemistry and the **Howard Hughes Medical Institute Laboratory, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Histones specifically phosphorylated at either tyro- sine (phospho-Tyr-histones) or serine and threonine (phospho-Ser-histones) were prepared and tested as substrates for so-called alkaline and protein phospha- tases. Three different preparations of alkaline phos- phatase (calf intestine, bovine liver, and Escherichia colg dephosphorylated phospho-Tyr-histones at 5-10 times the rate they dephosphorylated phospho-ser-his- tones. It was concluded that the p-nitrophenyl phos- phatase and phospho-Tyr phosphatase activities reside in the same protein on the basis of high performance liquid chromatography, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis, inhibition of both activ- ities by Pi and EDTA but not fluoride, and inhibition of phospho-Tyr phosphatase activity by p-nitrophenyl phosphate. Phosphoprotein phosphatase from rabbit muscle, which had little if any p-nitrophenyl phospha- tase activity, dephosphorylated phospho-Ser-histones at 16 times the rate it dephosphorylated phospho-Tyr- histones. Thus, the ratio of the pseudo-first order rate constants for dephosphorylation of phospho-Tyr-his- tones/phospho-Ser-histones was approximately 100 times higher for alkaline phosphatases compared to the rabbit muscle protein phosphatase. Membrane proteins from A-431 cells (phosphorylated at tyrosine) were more effective substrates than phospho-Tyr-histones as substrates for alkaline phosphatase; this was not the case for protein phosphatase. Phospho-Tyr phospha- tase activity of the alkaline phosphatases was optimal in the pH range of 7-8. The so-called alkaline phospha- tases, then, may be a group of membrane-bound gly- coproteins that represent a class of phosphoprotein phosphatases that show selectivity for proteins phos- phorylated at tyrosine residues.

Alkaline phosphatases from various mammalian tissues and bacteria have been studied extensively, but their physiological function remains unknown (1,2), even though a wide variety of low molecular weight phosphate esters can serve as sub- strates (1, 2).

Earlier reports from various laboratories showed that puri- fied preparations of phosphoprotein phosphatases had little, if any, activity toward PNPP’ as substrate (3-7). Phosphopro-

* This work was supported by National Institute of Health Grants HD10254 and HD00700. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 Research Professor of the American Cancer Society. ’ The abbreviations used are: PNPP, p-nitrophenyl phosphate; phospho-Tyr-histone, histones phosphorylated at tyrosine residues;

tein phosphatase activity in some tissues, however, is closely associated withp-nitrophenyl phosphatase activity (8,9), and it remains unclear whetherp-nitrophenyl phosphatase activity associated with phosphoprotein phosphatases is intrinsic or due to contamination by alkaline phosphatase (3, 8). PNPP has little structural resemblance to phosphoserine or phos- phothreonine (10, ll), but does resemble phosphotyrosine, a recently reported component of some phosphoproteins (12- 16). The structural resemblence of PNPP with phosphotyro- sine raised the possibility that the alkaline phosphatases might selectively dephosphorylate proteins phosphorylated at tyrosine residues. Such a possibility is particularly attractive since many or all mammalian alkaline phosphatases are mem- brane-bound glycoproteins and are thus localized in an organ- elle where tyrosine phosphorylation is known to occur (12- 16). This hypothesis was tested by using phospho-Tyr-his- tones, phospho-Ser-histones, and membrane proteins from A- 431 cells (phosphorylated at tyrosine) as substrates for alka- line phosphatases and phosphoprotein phosphatase. In this communication we show that alkaline phosphatase prepara- tions from calf intestine, bovine liver, and Escherichia coli selectively dephosphorylate phospho-Tyr-histones and A-431 membrane proteins at low enzyme concentrations, whereas phosphoprotein phosphatase from rabbit muscle selectively dephosphorylates phospho-Ser-histones.

EXPERIMENTAL PROCEDURES

Materials-Histone type IIA, phosphoserine, phosphothreonine, alkaline phosphatases from calf intestine (type VII), bovine liver (type IX), E . coli (type 111), PNPP, and protease from Streptomyces griseus (type VI) were obtained from Sigma. 0-Phosphotyrosine was prepared by a published procedure (17). Epidermal growth factor and membranes from A-431 cells were prepared as described previously (18, 19). Purified cyclic AMP-dependent protein kinase (catalytic subunit) and partially purified phosphoprotein phosphatase from rabbit muscle were kindly provided by Dr. Balwant S. Khatra of the Physiology Department, Vanderbilt Medical Center.

Assays-Phosphoprotein phosphatase activity with phospho-Tyr- histone, phospho-Ser-histone, or A-431 membrane proteins as sub- strate was assayed at 30 “C in a total volume of 50 pl containing 50 m~ Tris/HCl, pH 7.2, 1 mM dithiothreitol, 100 m~ NaCI, and the indicated amount of substrate. After incubation with the enzyme at 30 “C for 10 min, the reaction was stopped by the addition of 150 pl of 1.44% silicotungstic acid in 0.11 M H2S04 followed by 20 p1 of 20 mg/ml of bovine serum albumin. After standing 15 min at 4 “C, the suspension was centrifuged and 120 pl of clear supernatant fluid was assayed for radioactivity in a liquid scintillation spectrometer. Tris buffer was replaced by an appropriate buffer when measuring the pH dependence of enzyme activity.

phospho-Ser-histone, histones phosphorylated at serine and threo- nine; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate.

8197

8198 Phosphotyrosine Phosphatase Activity of Alkaline Phosphatase

p-Nitrophenyl phosphatase activity was assayed at 30 "C (total volume of 2.0 ml) in a solution containing 50 m~ glycine buffer a t pH 9.5 and 5 m~ PNPP. After incubation for 2 min the reaction was stopped by the addition of 1.0 ml of 2 M Na~C03 solution. The amount of product formed was estimated at 430 nm.

Preparation of Phosphorylated Histones-Phosphorylation of his- tones at serine and threonine was carried out using the catalytic subunit of the cyclic AMP-dependent protein kinase. The reaction mixture for histones, in a total volume of 4.0 ml, contained 40 mg of histones type IIA, 200 p~ [y3'P]ATP (6.8 X 10' cpm), 1 m~ dithio- threitol, 25 n" MgCL, 50 m~ Tris-HC1, pH 7.5, 50 units of protein kinase. After incubation at 22-24 "C for 20 h, histones were precipi- tated with 25% trichloroacetic acid, collected by centrifugation, and resuspended in water. The suspension was dialyzed against 2 X 500 ml of 25 mM 2-(N-morpholino)ethanesulfonate buffer a t pH 7.0 and finally dialyzed against 500 ml of 5 m~ Tris buffer, pH 7.2.

Phosphorylation of histones or A-431 membrane proteins at tyro- sine residues was carried out by a modification of the procedure of Ushiro and Cohen (15). The reaction mixture for phosphorylation of histones contained 20 m~ 4-(2-hydroxyethyl)-l-piperazineethanesul- fonic acid buffer a t pH 7.5, 2 m~ MnC12, [y3'P]ATP (250 p ~ , 1.7 X 10' cpm), 10 m~ PNPP, 6 mg of histones type IIA, 2 pg of epidermal growth factor, and the A-431 membrane preparation (1.5 mg of protein) in a total volume of 1.0 ml. After incubation for 20 min at room temperature the reaction was terminated by the addition of 25% trichloroacetic acid; sodium pyrophosphate was then added to a final concentration of 10 m ~ . The precipitated proteins were collected by centrifugation. After one more precipitation the proteins were di- alyzed against 500 ml of 25 m~ 2-(N-morpholino)ethanesulfonate buffer, pH 7.0, and then dialyzed against 5 mM Tris buffer at pH 7.2. After dialysis the suspension was centrifuged to remove membranes and the supernatant fluid containing histones was used for the assays. Membrane proteins of A-431 cells were phosphorylated essentially by the same procedure.

The amount of protein labeled with 32P and the amount of phos- phate released from the substrates were calculated from the specific activity of the radioactive ATP used as a precursor in the protein phosphorylation reaction.

Identification of the Phosphorylated Residue-Of phosphohis- tones, 100 pg were incubated with an equal amount of a protease from S. griseus in 50 mM Tris-C1 buffer, pH 7.5, for 20 h at 20-24 "C or were hydrolyzed with 6 N HCl under vacuum at 100 "C for 4 h. Thin layer chromatography (cellulose MN 300-coated plates) of the pro- tease digest or the acid hydrolysate was carried out in 1-butanol/ isopropyl alcohol/formic acid/water (3:1:1:1, v/v). Standards were detected by spraying with ninhydrin (0.2% in acetone).

Separation of Phosphorylated Amino Acids by HPLC-Separa- tion of phosphoserine, phosphothreonine, and phosphotyrosine by HPLC was carried out on a Beckman Ultrasil Ax anion exchange column (0.46 X 25 cm). The column was equilibrated with 15 mM potassium phosphate buffer, pH 3.8. Elution was carried out with the equilibration buffer for 25 min at a flow rate of 2.0 ml/min after the injection of 100-200 pl of sample. Fractions of 0.3 min were collected. In all cases, nonradioactive standards were added and detected by recording the absorbance at 206 nm, while 32P was detected by counting the samples for radioactivity in a liquid scintillation counter.

RESULTS

Dephosphorylation of Phosphoproteins by Alkaline and Protein Phosphatases-Two different types of phosphoryl- a ted histones were prepared by using specific kinase prepa- rations known to phosphorylate at different amino acid resi- dues. SDS-polyacrylamide gel electrophoresis of phospho- Tyr-histones showed very little contamination ( ~ 5 % ) by pro- teins originally associated with the A-431 membranes used for the preparation of phospho-Tyr-histones. Furthermore, an autoradiograph of the SDS-gel showed no radioactivity asso- ciated with the contaminating membrane proteins (data not shown). A thin layer chromatograph of a pronase digest or acid hydrolysate of phospho-Ser-histones showed phospho- serine and phosphothreonine and no phosphotyrosine as com- ponents, whereas phospho-Tyr-histones showed phosphoty- rosine as the only phosphorylated residue. The identity of the phosphorylated residues was also c o n f i i e d b y HPLC (Fig. 1). Using these substrates, alkaline phosphatase preparations

I A P-Ser + 1200 -

BOO - - E,

400- V

> k 1 5 a 2 0 40 60

e n a [L

I I 1

B P-Tyr I 5 0

I P-mr _ _ I 80

FRACTION NUMBER

FIG. 1. Separation of phosphotyrosine, phosphoserine, and phosphothreonine by HPLC on an Ultrasil Ax anion exchange column. Details of the method are given under "Experimental Pro- cedures." A, acid hydrolysate of phospho-Ser-histones; B, pronase digest of phospho-Tyr-histones.

TABLE I Phospho-Tyr-histone and phospho-Ser-histone phosphatase

activitv of various DhosDhatases

Enzyme source

Alkaline phospha- tases

Calf intestine Beef liver E. coli

Phosphoprotein phosphatases

Rabbit muscle

Substrate Activity ( vm/

Phospho- Phospho- ratio

K,)Tyr/ Tyr-his- Ser-his- cLz$l (Vm/K,)Ser'

tone" tone" pmol/min

1.06 0.20 5.3 11.7 0.80 0.10 8.0 18.0 0.29 0.05 5.8 12.8

0.42 6.84 0.06 0.13

a Phospho-Tyr-histone phosphatase activity was measured at a substrate concentration of 0.56 p~ (32Pi), and phospho-Ser-histone phosphatase activity was measured at a substrate concentration of 1.36 p~ (32Pi). ' 32P-Tyr/32P-Ser is the ratio of activity with phospho-Tyr-histone and phospho-Ser-histone as substrate.

e (V,,,/K,)Tyr is the ratio of maximum velocity to Michaelis con- stant with phospho-Tyr-histone as substrate; (V,,,/K,)Ser is the same ratio with phospho-Ser-histone as substrate. VJK,,, ratio was esti- mated from the slope of the u versus [a plots a t substrate concentra- tion where the velocity is a linear function of the substrate concen- tration.

from three different sources were shown to dephosphorylate phospho-Tyr-histones at a much higher rate than phospho- Ser-histones (Table I). Phospho-Ser-histone, in contrast, was the preferred substrate for phosphoprotein phosphatase from rabbit muscle. These observations suggested that the so-called alkaline phosphatases represent a group of phosphoprotein phosphatases with selectivity for phosphotyrosine residues. In this respect it is relevant to point out that the membrane

Phosphotyrosine Phosphatase Activity of Alkaline Phosphatase 8 199

preparations from A-431 cells have high p-nitrophenyl phos- phatase activity, and that these membrane preparations also have high protein phosphatase activity toward endogenous phosphoproteins (20), proteins known to be phosphorylated at tyrosine residues (15). Due to the high endogenous phos- phatase activity of these membrane preparations, it was not possible to prepare sufficiently high concentrations of phos- pho-Tyr-histones to directly estimate the K,. Therefore, Vm/ K, values were estimated from the slope of v versus [SI plots at substrate concentrations where velocity was a nearly linear function of substrate concentration. This ratio represents the pseudo-first order rate of dephosphorylation. As compared to

I A p Phospho-Ser-Histones . I

2 0 0 0 1 p

1 I I I

N * 3000- ’ /x Ii-

0 A 4 3 1 Membrane Proteins-

I O 0 0 I’ n I I

S 10 IS 20

.a

TIME (mln)

FIG. 2. Dephosphorylation of phospho-Ser-histones and A- 431 membrane proteins by alkaline and protein phosphatases. A, phospho-Ser-histones (14 p ~ , 32P, 2 mg/ml of histone) as substrate; B, phosphorylated proteins from A-431 membranes (0.1 p~ 32Pi, 0.3 mg/ml of protein) as substrate. 0.25 pg of calf intestinal alkaline phosphatase (X---X) and 0.5 pg of rabbit muscle protein phosphatase (M) were added in each assay.

I I 1

B

25 35 45

. I

.05

skeletal muscle phosphoprotein phosphatase, alkaline phos- phatases showed high selectivity for phospho-Tyr-histones as substrate (about 100 times higher (Vm/Km)Tyr/( V,/K,)Ser ratio, Table I).

When phosphorylated histone f2b was used as the substrate, instead of mixed histones, qualitatively similar results were obtained (data not shown).

Since it was possible that alkaline phosphatases showed selectivity only with histones as substrates, membrane pro- teins from A-431 cells were phosphorylated at tyrosine resi- dues and compared with phospho-Ser-histones as substrates. As can be seen in Fig. 2, the alkaline phosphatase also showed very high selectivity for A-431 membrane proteins phospho- rylated at tyrosine. The apparent initial rate of dephosphor- ylation, in fact, was more rapid with the membrane proteins than with phospho-Tyr-histones.

Alkaline Phosphatases Remove P, from Phosphorylated Proteins-It was possible that alkaline phosphatase prepara- tions contained protease activity which would produce acid- soluble 32P-labeled peptides from phosphohistones. Therefore, phospho-Tyr-histones were incubated at 30 “C for 30 min with alkaline phosphatases (calf intestine, bovine liver, and E. coli) and the product was analyzed by TLC and HPLC as described under “Experimental Procedures.” The radioactive product was identified as 32Pi based on its co-migration on TLC and its co-elution on HPLC with standard 32Pi. No other radioactive products were observed. Similar results were ob- tained when 32P-labeled A-431 membrane proteins were in- cubated with calf intestinal alkaline phosphatase. These re- sults suggest that the alkaline phosphatase preparations used dephosphorylate the indicated substrates and that proteases do not contribute to any of the observed activity.

Purity ofAlkaline Phosphatase Preparations-Various ex- periments were designed to evaluate the purity of the alkaline phosphatase preparations used in these studies since it was possible that p-nitrophenyl phosphatase activity and phos- pho-Tyr-histone phosphatase activity actually resided in dif- ferent proteins. High performance liquid chromatography of alkaline phosphatase preparations showed that phospho-Tyr- phosphatase activity and p-nitrophenyl phosphatase activity comigrated in all three cases (Fig. 3). SDS-polyacrylamide gel electrophoresis of calf intestinal or of E. coli enzyme prepa- rations also showed only one protein-staining band (Fig. 4). The liver alkaline phosphatase, however, was not homogene- ous on SDS-gels.

In addition, PNPP was an inhibitor of the phospho-Tyr- histone phosphatase activity of intestinal alkaline phospha- tase (Table 11). NaF, however, a known inhibitor of other

FIG. 3. High performance liquid chromatography of alkaline phos- phatases on a TSK 3000 SW column. The column was equilibrated with 25 mM triethanolamine buffer at pH 7.8 con- taining 1 m~ MgC12. The enzyme solu- tion (200 11.1) was injected and the elution was carried out with the same buffer at a flow rate of 1.0 ml/min. 0.5-ml fractions were collected. Fractions were assayed for p-nitrophenyl phosphatase activ- ity ( O ” 3 ) and phospho-Tyr-histone phosphatase activity (X---X). A, aka- line phosphatase from calf intestine; B, from bovine liver; C, from E. coli.

FRACTION NUMBER

8200 Phosphotyrosine Phosphatase Activity of Alkaline Phosphatase

phosphoprotein phosphatases (21, 22) did not inhibit either phospho-Tyr-phosphatase or p-nitrophenyl phosphatase ac- tivity (Table 11). EDTA completely inhibited both of these activities. Inorganic phosphate, a known inhibitor of intestinal alkaline phosphatase (23), inhibited the phospho-Tyr-phos- phatase activity of alkaline phosphatase at micromolar con- centrations (Table 11). These observations suggest that p- nitrophenyl phosphatase and phospho-Tyr-phosphatase ac- tivities are associated with the same protein.

p H Optima-Phospho-Tyr-histone phosphatase activity

A B FIG. 4. SDS-polyacrylamide gel electrophoresis of ( A ) calf

intestinal alkaline phosphatase and (B) E. coli alkaline phos- phatase. The electrophoresis was carried out in 7.5% polyacrylamide gels at pH 8.8 in the presence of 0.1% SDS as described in (24).

TABLE I1 Effect of inhibitors on the phospho-Tyr-histone phosphatase

activity of alkaline phosphatase-from calf intestine Additions Relative activity

None PNPP

2 mM 5 mM

Pi 20 PM

200 PM 10 mM

EDTA, 20 mM (10-min preincubation) Sodium fluoride. 50 mM

s 1 0 0

24 12

82 66 26 3

92

0

O x R

f 1.2 x *

0 X n

- 1 ” 0

1.0 a

w 0.8 a

W -1

FIG. 5. Effect of pH on phospho-Tyr-histone phosphatase activity of calf intestinal alkaline phosphatase. The activity was determined at 0.1 PM substrate (‘”P,) as described under “Experimen- t a l Procedures” except that different buffers at different pH values were used. 2-(N-Morpholino)ethanesulfonate buffer, pH 5.5-7.0 (0); Tris-CI, pH 7.0-8.5 (x); 4-(2-hydroxyethyl)-l-piperazineethane sulfo- nate buffer, pH 7.0-8.0 (0); glycine-NaOH, pH 8.5-9.5 (0).

was optimal over a broad range with apparent peak activities at pH 7.0 and 8.0 (Fig. 5). These two pH optima, however, could be due to buffer effects. These pH optima are much lower than when high concentrations of PNPP are used (23). The pH optimum close to neutral pH is suggestive of physio- logical significance of the phospho-Tyr-histone phosphatase activity of the alkaline phosphatases.

DISCUSSION

From the results presented here it is clear that alkaline phosphatase preparations can selectively dephosphorylate various proteins phosphorylated at tyrosine. The purity of calf intestinal and E. coli alkaline phosphatases and the effect of inhibitors on phosphotyrosine phosphatase activity suggest that phosphotyrosine phosphatase activity is the intrinsic property of alkaline phosphatases. This is not surprising since phosphotyrosine is structurally similar to PNPP and phenyl phosphate, excellent substrates for alkaline phosphatases.

Is the dephosphorylation of phosphotyrosine-containing proteins by alkaline phosphatases physiologically significant? The following observations suggest physiological significance of the observed selectivity: (a) membrane proteins from A-431 cells, natural substrates, are very good substrates; ( b ) A-431 membrane preparations contain bothp-nitrophenyl phospha- tase and protein phosphatase activity toward endogenous phosphoproteins, proteins known to be phosphorylated at tyrosine; (c) the pH optima for the phosphotyrosine phospha- tase activity of alkaline phosphatase is close to physiological pH; (d) the concentration of alkaline phosphatase used was relatively low, generally between 5 and 40 m; and (e) epider- mal growth factor is known to decrease alkaline phosphatase in HeLa and JEG (choriocarcinoma) cells (25).

The possible protein phosphatase function of alkaline phos- phatase has been explored in a number of previous studies (8, 26, 27). Alkaline phosphatase from E. coli, for example, is known to dephosphorylate glycogen synthase D, phosphory- lase kinase, and phosphohistone but not glycogen phosphor- ylase (26). The high concentrations (equimolar enzyme and substrate) of alkaline phosphatase used, however, led these authors to conclude that the protein phosphatase activity of the E. coli enzyme was probably of little physiological signif- icance. A purified preparation of alkaline phosphatase from human placenta, in contrast, was shown to dephosphorylate histones, protamine, glycogen synthase, casein, and phosvitin at lower alkaline phosphatase concentrations (27); this alka- line phosphatase also did not dephosphorylate phosphorylase

Phosphotyrosine Phosphatase Activity of Alkaline Phosphatase 8201

a. Li et al. (8) have suggested that the association of alkaline phosphatase activity with phosphoprotein phosphatases of M, = 35,000 is a general phenomenon and that alkaline phospha- tase activity is involved in the regulation of protein phosphor- ylation-dephosphorylation reactions. From the results pre- sented in this paper it can be suggested that alkaline phos- phatases may be involved in the regulation of phosphoryla- tion-dephosphorylation of those proteins (especially mem- brane proteins) which are phosphorylated at tyrosine residues; this does not preclude the possibility that alkaline phospha- tases also function to dephosphorylate serine and/or threo- nine in membrane preparations.

Alkaline phosphatase activity is present in a wide variety of mammalian tissues such as intestinal mucosa, spleen, pla- centa, and liver (1). In addition, alkaline phosphatase activity is known to change dramatically in various disease states where altered cell growth or transformation occw (28-31). If the function of the alkaline phosphatases is to dephosphory- late phosphotyrosine-contg proteins, one would expect both alkaline phosphatase and protein kinases capable of phosphorylating tyrosine to be present in these same tissues. Hunter and Sefton (13) have suggested, in fact, that all ver- tebrate cells contain at least one protein kinase (the product of sarc gene) that can phosphorylate at tyrosine.

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