Protein tyrosine kinase and protein phosphotyrosine phosphatase in normal and psoriatic skin

Biochimica et Biophysica Acta,

798 (1984) 53-59

53

Elsevier BBA 21690

PROTEIN TYROSINE KINASE AND PROTEIN P H O S P H O T Y R O S I N E PHOSPHATASE IN NORMAL AND PSORIATIC SKIN SUSAN GENTLEMANa, TODD M. MARTENSENb, JOHN J. DIGIOVANNA c and GERALDJ. CHADER a a Laboratory of Vision Research, National Eye Institute, b Laboratory of Biochemistry, National Heart, Lung and Blood Institute and c DermatoloKv Branch, National Cancer Institute, National Institutes of Health, Bethesda, AID 20205 (U.S.A.)

(Received August4th, 1983)

Key words: Tyrosine kinase; Protein phosphatase; Psoriasis," (Skin)

Protein tyrosine kinase and protein phosphotyrosine phosphatase activities were measured in extracts of skin samples from patients with psoriasis. Both kinase and phosphatase activities were significantly greater in samples taken from an involved area, characterized by epidermal hyperproliferation, than from adjacent skin of normal appearance. Samples from skin of non-psoriatic individuals were indistinguishable from the normal-appearing skin of psoriatic patients. There was no detectable change in the apparent K m for either ATP or casein of the protein tyrosine activity in plaques compared with controls. Phosphorylation of endogenous proteins was also increased about 2-fold in plaque extracts compared with controls. Both epidermal growth factor and platelet-derived growth factor stimulated endogenous protein tyrosine phusphorylation in particulate fractions of plaque biopsies but not in solubilized extracts nor in any control fractions. Our data suggest that increased protein tyrosine phosphorylation and dephosphorylation activity and growth factor sensitivity are important factors in non-malignant hyperplastic cell growth.

Introduction The discovery of phosphotyrosine residues and protein kinase activity which was tyrosine-specific in the Rous sarcoma virus transforming protein pp60src suggested that protein tyrosine phosphorylation was important in transformation [1]. Further studies have shown tyrosine-specific protein kinase activity not only intrinsic to several viral transforming proteins but also associated with the receptors of several rnitogenic growth factors [2,3]. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), both necessary to initiate DNA synthesis in cultured fibroblasts, Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate. 0304-4165/84/$03.00 © 1984 ElsevierSciencePublishers B.V.

stimulate protein tyrosine kinase activity and tyrosine phosphorylation of their receptors in fibroblasts [4]. Protein tyrosine kinase activity is also associated with growth factor receptors and insulin receptors in several other tissues [5-7]. Thus, increased protein tyrosine kinase activity appears to be an early event correlating with processes leading to accelerated cell proliferation. EGF activation of the receptor-associated kinase activity, however, was found to be insufficient to promote mitogenesis [8], and EGF receptor number and EGF stimulation of phosphorylation has been reported to be severely decreased in regenerating rat liver [9]. Moreover, no significant increase in protein phosphotyrosine was observed in rapidly growing chick cells except in retrovirustransformed cells [10]. In contrast, tyrosine dephosphorylation activity is as yet poorly characterized, although phosphotyrosine protein phos-

54 phatase activity has been found in both soluble and particulate fractions from several cell lines and tissues [11,12]. Since the phosphorylation state of tyrosine residues is a result of the relative activities of kinase(s) and phosphatase(s), it is desirable to study both activities in a tissue to determine their involvement in cellular regulation and the importance of tyrosine phosphorylation in relation to growth factor stimulation of cellular proliferation. Psoriasis is a disorder characterized by areas of epidermal, non-malignant hyperproliferation. Thick plaques of rapidly dividing epidermal cells are sharply demarcated from skin of normal appearance [13]. We thought this an excellent model system for studying non-transformed, cellular proliferation and, in the present study, report on characteristics of both the kinase and phosphatase activities in this hyperproliferative disease. Methods

Sample preparation. Punch biopsies were taken from the edge of a psoriatic plaque and from adjacent skin of normal appearance (psoriatic controls) in patients with active untreated plaque-type psoriasis. After local anesthesia with 1% xylocaine without epinephrine, 3 mm punch biopsies from six psoriatic patients were taken and frozen on dry ice. Punch biopsies from donors having no history of psoriasis were taken as normal controls. After local anesthesia with 1% xylocaine without epinephrine, 3 mm punch biopsies from one normal donor was taken and frozen on dry ice. Additional normal punch biopsies were obtained from the periphery of surgical specimens obtained under general anesthesia. Enzyme activities from normal controls were not affected by the type of anesthesia. The frozen samples were minced on dry ice, transferred to a glass-on-glass conical homogenizer and homogenized by hand in 0.5 ml of 25 mM Hepes buffer (pH 7.5) containing 25% (v/v) glycerol, 10 mM CHAPS detergent and 1 mM dithiothreitol. The homogenates were centrifuged at 27000 × g for 20 rain. The supernatant fractions were removed and reserved. Protein concentrations ranged from 0.5 to 1.5 mg/ml. All fractions were stored at - 2 0 ° C until assayed. Protein tyrosine kinase activity was stable for at least two months under these conditions.

Assay. Protein tyrosine kinase activity was assayed by quantitative determination of [32p]phosphotyrosine in protein after base hydrolysis and ion exchange chromatography [14]. The reaction medium contained 25 mM Hepes buffer (pH 7.5), 10 mM MgC12, 5 mM MnC12, 1 mM dithiothreitol, 1-50/~M [y-32p]ATP (2-5 ktCi/assay), 0.1 0.2 m g / m l enzyme protein, 2 m g / m l casein and 10 mM CHAPS in a final volume of 50 /~1. The reaction was started by addition of enzyme, incubated for 5 min at 37°C and stopped by addition of 0.5 ml of 20% (w/v) trichloroacetic acid. The precipitate was collected by centrifugation and hydrolyzed for 30 rain at 155°C in 100/~1 of 5 N KOH containing 1 /~M [14C]phosphotyrosine (17000 cpm/assay) for determination of recovery. The hydrolysate was applied to a 0.5 ml Bio-Rad AG-50 column (H + form, 200-400 mesh) and washed with 3 ml of 0.1 N formic acid containing 1% H3PO 4. Phosphotyrosine was then eluted with three 1 ml distilled water rinses. The two peak fractions were each taken up in 10 ml scintillation fluid and the radioactivity determined. A constant 14C/32 p ratio, indicating separation of phosphotyrosine from inorganic phosphate, was routinely obtained in these fractions. Protein phosphotyrosine phosphatase activity was determined by the release of 32Pi from [32 P]phosphotyrosyl glutamine synthetase [15]. The reaction medium, containing 25 mM Hepes buffer (pH 7.0), 1 mM dithiothreitol, 12.6 ~M [32p]phosphotyrosyl glutamine synthetase (175 000 cpm) and 0.6 m g / m l enzyme protein in 60 ~1, was incubated for 5 min at 37°C and stopped by addition of 0.1 N HC1 containing 5 m g / m l bovine serum albumin. The protein was precipitated with 0.5 ml of 10% trichloroacetic acid, centrifuged, and an aliquot of the supernatant fraction was counted. 97% of the 32p in the acid supernatant was extractable by molybdate in butanol/benzene (1:1), indicating that it was inorganic phosphate rather than soluble phosphopeptides. All assays were carried out in duplicate with extracts from at least two donors. Protein concentrations were determined by the method of Bradford [16] using bovine serum albumin as the standard. DNA content was measured by the method of Kissane and Robbins [17] using salmon sperm as the standard.

55

Materials. [y-32p]ATP was obtained from New England Nuclear and [t4C]tyrosine from Amersham. Casein (5% solution, partially hydrolyzed) was from Sigma and CHAPS detergent from Calbiochem. All other chemicals were reagent grade from standard sources.

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Results

I 5

The specificity of the kinase assay for formation of [32p]phosphotyrosine was demonstrated by amino acid analysis (Fig. 1). The water-eluted AG-50 fractions from assays of two preparations were pooled and chromatographed on an amino acid analyzer as previously described [14]. More than 80% of the 32p activity in the water-eluted peak co-chromatographed with authentic [14C]phosphotyrosine. The trace of inorganic phosphate could be eliminated entirely by discarding the first 0.5 ml of the water-eluted material. Zero time and reagent blanks contained a total of less than 25 cpm of 32p in the [14C]phosphotyrosine peak from AG-50 columns. Dependence of protein tyrosine kinase and phosphatase activities on time and enzyme concentration is shown in Fig. 2. Protein tyrosine kinase activity was approximately linear with time

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Fig. 1. Chromatography of putative phosphotyrosine peak eluted from AG-50 on calibrated Dionex analyzer. Detergentsolubilized extracts of human skin were incubated with [32p]ATP and casein. The water-eluted fractions from the AG-50 column were pooled and 50% of the volume was taken for liquid scintillation counting. The remaining volume was lyophilized and taken up in 125 #gl of 0.2 M sodium formate (pH 2). 50 #1 of this volume was applied to a Dionex column calibrated with authentic phosphotyrosine, e, 32p; © [ 14C]tyrosine phosphate.

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Fig. 2. Dependence of protein tyrosine phosphorylation on time of incubation and protein concentration of tissue extract. Detergent-solubilized extracts of human skin were incubated for varying times and at varying extract protein concentrations. A, B: protein tyrosine kinase activity assayed with 15 /xM [32p]ATP. C, D: protein phosphotyrosine phosphatase activity.

to 5 rain and with protein to 0.3 mg/ml under these assay conditions. Protein phosphotyrosine phosphatase activity was linear with time to 10 min and with protein concentration to at least 0.1 mg/ml under these assay conditions. As shown in Table I, both enzymatic activities were significantly increased in extracts from the plaque samples compared with the paired control samples from the same patients or nonpsoriatic controls. Protein tyrosine kinase activity increased approx. 3.5-fold and protein phosphotyrosine phosphatase activity approx. 2.5-fold in these extracts. In the absence of detergent, protein tyrosine kinase activity was associated primarily with the particulate fraction. Assay of the kinase activity of the particulate fraction prepared without detergent also showed a 3-fold increase in samples from plaque areas (data not shown). Thus, differential detergent solubilization or activation in the plaque versus control samples is not a factor in the elevated enzymatic activities observed. Endogenous phosphorylation activity was also increased

56 TABLE I P R O T E I N TYROS1NE K I N A S E ACTIVITY IN D E T E R G E N T - S O L U B I L I Z E D EXTRACTS OF N O R M A L A N D PSORIATIC SKIN Detergenl-solubilized extracts of punch biopsies were prepared and assayed for protein tyrosine kinase activity. The substrate concentrations were 20/zM [ 32P]ATP and 2 m g / m l casein. Extract protein concentration in the reaction ranged between 0.15 and 0.26 mg/ml. Extract

Protein tyrosine kinase (pmol [ 32 P]phosphotyrosine/ mg per min, mean + S.E.) Endogenous

+ Casein

Normal control Psoriatic control Psoriatic plaque

0.3 + 0,3 0.4 + 0.2 0.8 + 0.5 a

0.7 _+0.4 0.7 + 0.3 2.4 + 1.1 b

Protein phosphotyrosine phosphatase (nmol P i / m g per min, mean + S.E.)

n

-0.43 + 0.12 1.04 _+0.24 b

4 6 6

a p ~ 0.025, b p ~ 0.005, paired difference t-test compared to either control.

approx. 2-fold in the psoriatic plaque compared with either control, which may indicate an increased availability of protein substrate a n d / o r reflect the change in kinase specific activity observed with the exogenous substrate. Since the psoriatic plaque has a thicker epidermis and thus contains a larger number of epidermal cells per unit area than the uninvolved adjacent skin, the higher enzymatic activities might be due simply to the increase in cell number. However, comparison of DNA content (as a measure of cell number) and the protein tyrosine kinase and protein phosphotyrosine phosphatase activities do not support this interpretation (Table II). Whether the enzymatic activities were expressed on the

basis of protein content or DNA content of the samples, higher activities of the plaque samples compared with the controls were always observed. The protein tyrosine kinase activity was proportional to casein concentration, approaching saturation at 2 m g / m l (Fig. 3). Maximal activity was 3-4-fold higher in psoriatic extracts than in control extracts but both exhibited approximately the same apparent g m for casein. Likewise, the apparent K m for ATP was about 10/~M ( + 2, n = 5) in both psoriatic and control extracts, but the maximal activity was 3-fold greater in the psoriatic plaque extract (Fig. 4). There was no difference in the apparent K m for ATP with either casein or endogenous protein as the phosphate acceptor or

T A B L E II C O R R E L A T I O N O F P R O T E I N T Y R O S I N E K I N A S E A N D P R O T E I N P H O S P H O T Y R O S I N E P H O S P H A T A S E ACTIVITIES W I T H S O L U B L E P R O T E I N A N D D N A IN P L A Q U E A N D C O N T R O L BIOPSIES Enzymatic activity was measured as described in Table I. Protein (mg)

DNA (mg)

Protein tyrosine kinase

Protein phosphotyrosine phosphatase

pmol phosphotyrosine/mg protein per min

pmol phosphotyrosine/mg D N A per min

nmol P i / m g protein per min

nmol P i / m g D N A per min

Patient 1 Control Plaque

0.45 0.63

1.6 2.2

1.0 4.6

0.28 1.31

0.40 1.48

0.11 0.42

Patient 2 Control Plaque

0.41 0.50

0.5 1.2

0.3 1.2

0.22 0.54

0.16 0.54

0.14 0.22

57

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Fig. 3. Dependence of protein tyrosine kinase activity on casein concentration in detergent-solubilized extracts of psoriatic skin. Extracts were prepared from punch biopsies of psoriatic plaque and of adjacent uninvolved skin and assayed for protein tyrosine kinase activity, [32P]ATP concentration was 15 /~M. e, uninvolved control; ©, psoriatic plaque.

To determine whether growth factors might be involved in the increased protein tyrosine kinase activity of the psoriatic plaque, EGF was incubated with the detergent-extracted preparations. No effect on the kinase activity was observed. However, since detergent solubilization may disrupt growth factor-promoted phosphorylation [4,5], particulate fractions were prepared without CHAPS detergent. These fractions retained about 85% of the total protein tyrosine kinase activity and all of the endogenous phosphorylation. Stimulation of endogenous phosphorylation by both EGF and PDGF was observed only in the fractions from psoriatic plaque (Table III). Half-maximal stimulation by EGF was obtained at about 10 ng/ml EGF, and stimulation was maximal between 0.1 and 1 /~g/ml (Fig. 5). This range of effective EGF concentrations is comparable to that for EGF receptor binding and EGF-induced receptor down-regulation [5,18]. In contrast, particulate fractions from both normal and psoriatic control samples showed no detectable increase in

in extracts from nonpsoriatic controls compared with those from the uninvolved psoriatic controls (data not shown). E 2.5 E

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Fig. 4. Dependence of protein tyrosine kinase activity on ATP concentration in detergent-solubil~.~l extracts of psoriatic skin. Extracts were prepared and assayed as described in Fig. 3. Casein concentration was 2 mg/ml. O, uninvolved control; ©, psoriatic plaque.

oL--#

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101

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Fig. 5. EGF concentration dependence of endogenous protein tyrosine phospborylation in particulate fractions of normal and psoriatic skin. 27000x g particulate fractions of skin biopsies from normal and psoriatic donors were prepared as described in Table If. Phosphorylation of endogenous proteins was carried out in the absence of casein. [32p]ATP concentration was 20/~M. I , uninvolved psoriatic control; O, psoriatic plaque; z~, A, nonpsoriatic controls.

58 TABLE lll EFFECT OF EGF A N D P D G F ON E N D O G E N O U S PROTEIN A N D CASEIN TYROSINE PHOSPHORYLATION IN PARTICULATE FRACTIONS OF N O R M A L A N D PSORIATIC SKIN Punch biopsies weqre homogenized without detergent and centrifuged at 27000× g for 20 min. The pellet was resuspended in detergent-free buffer and assayed. Substrate concentrations were 5 /~M [32P]ATP and 2 m g / m l casein. Extract protein was 0.2 m g / m l . EGF concentration was 1 t~g/ml and P D G F was 0.1 ~ g / m l . Values are means of duplicate determinations that varied by no more than + 0.1 p m o l / m g per min ( _+0.02, mean + S.E.) Extract

Protein tyrosine phosphorylation (pmol [32 P]phosphotyrosine/mg per min) Endogenous

Normal 1 Normal 2 Psoriatic 1 Control Plaque Psoriatic 2 Control Plaque

+ Casein

Basal

+ EGF

+ PDGF

Basal

+ EGF

0.8 1.5 1.3 1.0 1.2 2.6

0.8 1.5 1.4 1.6 1.0 3.6

0.9 1.6 1.2 1.4 1.1 3.1

2.2 -4.1 5.4

2.2 -4.1 6.0

5.9

5.9

endogenous phosphorylation in the presence of the growth factors. Discussion

These results provide evidence for the importance of phosphotyrosine proteins in nontransformed cell proliferation in vivo. The 3.5- and 2.5-fold increases in specific activities of protein tyrosine kinase and protein phosphotyrosine phosphatase, respectively, in the psoriatic plaque correspond well with a reported 2.7-fold increase in mitotic index in psoriatic tissue [13]. Thus, these enzymes appear to be primarily associated with the dividing epidermal cells. It could be either that the specific activity of these enzymes is elevated in all cells in the plaque or that a subpopulation of cells with high specific activity is enriched in the plaque area. The coordinated increase of both kinase and phosphatase activities in these samples suggests the rate of turnover of phosphorylation of tyrosine residues in proteins may be more important than a simple increase in protein tyrosine phosphorylation in regulating cell growth. Our data show that the activity of the protein phosphotyrosine phosphatase is about two orders of magnitude greater than that of the protein tyrosine kinase in skin extracts. If these activities reflect the cellular activ-

ities, the phosphatase(s) must exert a substantial effect on protein tyr0sine phosphate levels and thus both activities must be considered in assessing the role of tyrosine phosphorylation in normal and abnormal proliferation. However, while the elevated levels of kinase and phosphatase activities may be involved in sustaining hyperproliferative growth in the psoriatic plaque, the basic pathogenetic defect may actually lie in an abnormal regulation of these enzymes by growth factors rather than in the enzymes themselves. The kinetic data show that the substrate affinities of the protein tyrosine kinase activity in the hyperproliferative plaque are not different from those of normal or uninvolved psoriatic skin. The change in maximal activity, but not apparent K m, for ATP and for casein demonstrates higher specific activity of protein tyrosine kinase due either to an increased amount of the kinase enzyme or to an increase in the activity "~of the existing kinase. We cannot distinguish between these two possibilities on the basis of the present results. Epidermal cells in the psoriatic plaque are well known to have a higher [3H]thymidine-labeling index [13,19], which could be related to the increased protein tyrosine kinase activity. Besides the major quantitative difference in enzyme activity in psoriatic vs. control tissue, two important qualitative differences are observed.

59 First, the activity is growth-factor sensitive in the plaque area and secondly there is increased endogenous phosphorylation. The elevated basal phosphorylation may be due primarily to autophosphorylation of the protein tyrosine kinase, that is, an increase in concentration of endogenous substrates. The stimulation of endogenous phosphorylation by EGF and PDGF only in particulate fractions from psoriatic plaque could be due either to existence of receptors not present in normal epidermis or present at levels too low to be detected and thus also represent increased endogenous substrates. It is interesting to note, in contrast, that EGF-stimulated phosphorylation was found to be reduced in the only other in vivo cell growth model (regenerating rat liver) reported to date [9]. However, these data were obtained at a single time point after partial hepatectomy when the number of receptors was at a minimum. Therefore, it is not clear how these results compare with the situation in the hyperproliferative psoriatic plaque. The loss of EGF stimulation in detergentsolubilized extracts of psoriatic plaque is similar to that reported for human fibroblast [4] and mouse liver membranes [5]. This may be due to reversible disruption of the ligand binding site by the detergent and not to destruction of the receptor or its associated kinase activity. The lack of EGF stimultaion of casein phosphorylation by particulate fractions from psoriatic plaque can be compared to the lack of EGF-stimulated phosphorylation of exogenous substrates by mouse liver m e m b r a n e s [5] and to E G F - s t i m u l a t e d thiophosphorylation of synthetic peptides by human A431 epidermoid carcinoma cell membranes [20]. Thus, the characteristics of EGF-stimulated phosphorylation in psoriatic skin are similar to those reported from other sources. The precise role of tyrosine-specific phosphorylation in cellular proliferation is not known. It would appear from the present results, however, that protein tyrosine turnover rather than simply

increased kinase activity per se may be of critical importance in hyperproliferation and that regulation of the sensitivity and/or number of associated growth factor receptors is intimately involved. References 1 Hunter, T. and Sefton, B.M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1311-1315 2 Pike, L.J., Marquardt, H., Todaro, G.J., Gallis, B., Casnellie, J.E., Bornstein, P. and Krebs, E.G. (1982) J. Biol. Chem. 257, 1462-1463 3 Ushiro, H. and Cohen, S. (1980) J. Biol. Chem. 255, 8363-8365 4 Ek, B. and Heldin, C.-H. (1982) J. Biol. Chem. 257, 10486-10492 5 Cohen, S., Fava, R.A. and Sawyer, S.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6237-6241 6 Petruzzelli, L.M., Ganguly, S., Smith, C.J., Cobb, M.H., Rubin, C.S. and Rosen, O.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6792-6796 7 Zick, Y., Kasuga, M., Kahn, C.R. and Roth, J. (1983) J. Biol. Chem. 258, 75-80 8 Schreiber, A.B., Libermann, T.A., Lax, I., Yarden, Y. and Schlessinger, J. (1983) J. Biol. Chem. 258, 846-853 9 Rubin, R.A., O'Keefe, E.J. and Earp, H.S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 776-780 10 Sefton, B.M., Hunter, T., Beemon, K. and Eckhart, W. (1980) Cell 20, 807-816 11 Brautigan , D.L., Bornstein, P. and Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522 12 Fouikes, J.G., Erikson, E. and Erikson, R.L. (1983) J. Biol. Chem. 258, 431-438 13 Farber, E.M. and Van Scott, E.J. (1979) in Dermatology in General Medicine 2nd Edn. (Fitzpatrick, T.B., Eisen, A.Z., Wolff, K., Freedberg, I.M. and Austen, K.F., eds.), pp. 233-248, McGraw-Hill, New York 14 Martensen, T.M. (1982) J. Biol. Chem. 257, 9648-9652 15 Martensen, T.M. and Stadtman, E.R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6458-6460 16 Bradford, M.M. (1976) Anat. Biochem. 72, 248-254 17 Kissane, J.M. and Robins, E. (1958) J. Biol. Chem. 233, 184-188 18 Krupp, M.N., Connolly, D.T. and Lane, M.D. (1982) J. Biol. Chem. 257, 1489-1496 19 Weinstein, G. and Frost, P. (1968) J. Invest. Dermatol. 50, 254-259. 20 Cassel, D., Pike, L.J., Grant, G.A., Krebs, E.G. and Glaser, L. (1983) J, Biol. Chem. 258, 2945-2950