Arch Dermatol Res (1992): 284:111 113

9 Springer-Verlag 1992

Casein kinase II activity in psoriatic epidermis V. I. Mitev 1, L. Miteva 2, and A. Dourmishev a

Department of Biochemistry, Institute of Biomedical Sciences, Sofia, Bulgaria, 2 Institute of Dermatology and Venereology, Medical Academy, 1431 Sofia, Bulgaria Received August 26, 1991

Key words: Psoriasis - Casein kinase II - Epidermal growth - Ser/Thr protein kinases

Casein kinase I and II (CK I and CK II) are spontaneously active messenger-independent ubiquitous Ser/Thrspecific protein kinases. Their name is derived from their preference for casein and phosvitin as in vitro substrates. They can be differentiated from each other by a number of features: in contrast to CK I, CK II has a different elution profile on DEAE-cellulose and phosphocellulose, it utilizes GTP as phosphate donor, it is inhibited by heparin and 2,3-bisphosphoglycerate (2,3BFG) and phosphorylates RRREEETEEE, its specific synthetic substrate (for review see references 4 and 14). CK II is an important enzyme identified in all cell types and species examined and is concerned with signal transduction [6]. Considerable interest in CK II has arisen recently because its activity is stimulated significantly in response to several mitogens [insulin, epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) etc], it phosphorylates nuclear oncoproteins, and plays a role in the transduction of extracellular signals to the nucleus [7, 13]. Psoriasis is an excellent model system for studying non-transformed, cellular proliferation [2]. CK II activity has been shown to be present at high levels in keratinocytes [3]. Because of the considerable interest in the role of growth factors in psoriasis [12], we investigated CK II activity in both clinically affected and unaffected psoriatic epidermis, as well as in normal epidermis. The group of patients with normal skin comprised five men and five women of mean age 39 years (range, 21-53 years). The psoriatic group comprosed four men and four women of mean age 46 years (range, 29-60 years). Both lesional and non-affected epidermal samples

Correspondence to: Vanio I. Mitev, M.D., Ph.D., Unit6 de Differenciation Cellulaire, Batiment des Biotechnologies I.N.R.A., F-78 350 Jouy-en-Josas, France

were obtained from each patient. Epidermal samples from untreated psoriatic plaques (in the early stage of development) and non-affected skin were taken by dermo-epidermal separation using suction [5]. All samples were taken from the volar aspect of the forearm. The tissue samples were cut into pieces using scissors and mixed with five volumes of ice-cold buffer (Tris-HC1 20 mM, pH 7.4, 0.25 M sucrose, 5 m M MgC12 and 10 mM KC1). After homogenization and filtration, the homogenate was centrifuged for 60 min at 120 000 g at 4 ~ [11]. The supernatant was injected on to a Mono Q 5/5 column (Pharmacia Uppsala Sweden). Proteins were eluted over a 20 ml gradient fiom buffer A (20 m M Tris-HC1, pH 7.5) to buffer B (0.4 M NaC1, 20 m M Tris-HC1, pH 7.5) at a rate of 1 ml/min. Fractions of 1 ml were collected. The Mono Q fractions showing CK II activity were loaded on to phosphocellulose (Whatman P 11, Maidenstone, Kent, UK, pretreated according to the manufacturer's instructions and equilibrated in 50raM phosphate buffer, pH6.8). The column (1.6x 10cm) was washed with 20ml 0.25 M NaC1, 50 m M phosphate buffer, pH 6.8, at a rate of 60 ml/h. The P l l was eluted with a 2 • linear gradient (0.25 1.25 M NaC1) in phosphate buffer. Fractions of 0.5 ml were collected and assayed for CK II activity. Further purification of the enzyme was achieved by chromatography on a second Mono Q column under the same conditions. The resulting enzyme had high specific activity and the preparation was more than 85% pure, as shown by SDS-PAGE. The purified enzyme was stored in 0.4 M potassium phosphate and 0.02% NaN 3 at 4 ~ until used. Protein kinase activity was determined as previously described [8] with some modifications. Reaction media contained 5 0 r a m Tris-HC1, pH 7.4, 140mM KC1, 1 0 m M MgC12, 0.1 m M IT-32pIATP (500 cpm/pmol, specific activity 5000 Ci/mmol, Amersham) and the indicated amounts of enzyme preparation and subtrates. Protein concentration was determined according to the Lowry method. We tested the chromatographic profile on Mono Q (Fig. 1) and found two peaks of phosvitin (casein) kinase activity. The first one eluted at 100 m M NaC1. The enzyme preferred casein to phosvitin, and heparin, 2,3-

112 Table 1. Characterizationofthetwophosvitin (casein)kinase peaks from a Mono Q column with normal epidermal cytosol. Peak I (CK I) was measured as decribed with the substrate partially dephosphorylated casein(Sigma) and peak II (CK II) with phosvitin (Sigma) in the presence of 5 ~tg of enzyme preparation.

Casein 3.7 mg/ml

Peak I (CK I) Peak II ( C K II)

Phosvitin 2 mg/ml

RRREEETEEE was a generous gift from Dr. T. Barth, Institute of Organic Chemistry and Biochemistry, CSAV, CSFR. Each value shown represents the average of three determinations. Activity is given in nmol, min per mg

Heparin 6.7 nmol/1

2,3-Bisphos phoglycerate 2 retool/1

RRREEETEEE 1 retool/1

GTP 0.5 retool/1

7.5 • 0.3

3.2 • 0.4

7.5 + 0.4

7.5 • 0.6

-

7.4 • 0.5

8.4 _4- 0.6

17.5 + 0.7

1.44- 0.1

5.0 + 0.4

6.5 __+0.4

3.3 __+0.4

4/I/*II00 //

20

~ I 0.2

i"

/

/

/

/

I

t

m o\o

Purified CK II

50

Normal epidermis (1)

239 _+ 17

Uninvolved psoriatic epidermis (2)

277 _+ 24

116

(n = 5)

1426 _+ 31

596

(n = 3)

co

CK

0.'~

/

-g

/ /

Table 2. Activities of highly purified CK II preparations from different samples. Protein kinase activity was measured with phosvitin as substrate (2 mg/ml) and 1 gg of CK II preparation. The significance of the difference between the samples was assessed by Student's t-test

Psoriatic Plaques (3)

CK I ----~d / l /

Activity (nmol/min per rag)

Activation (%)

Significance

100 2 vs 1 P < 0.05

3 vs 1/2 P < 0.001

r.) ,-I

I

0.0 1234567

8 9 10 11 12 13 14 15 16 17 18 19 20

FRACTION

NUMBER

Fig. 1. Elution profile of CK I and CK II on a Mono Q column. Casein kinase II eluted at between 180 and 220 m M NaC1.

BFG and GTP did not affect its activity (Table 1). This chromatographic and biochemical profile corresponded to CK I. The second peak of enzyme activity eluted at 200 m M NaC1. This enzyme preferred phosvitin to casein and utilized GTP. The kinase phosphorylated the specific subtrate for CK II (RRREEETEEE) and was inhibited by heparin and 2,3-BFG. It corresponded to CK II. The Mono Q fractions showing CK II activity were loaded on to Pl 1 and CK II was eluted at about 0.7 M NaC1. After the second Mono Q column, highly purified CK II preparations from different samples were compared. As shown in Table 2, CK II activity from psoriatic plaque samples was 5.14 times higher than from unaffected psoriatic epidermal samples. The unaffected psoriatic epidermis showed a slightly higher activity than nonpsoriatic epidermis. These results correlate with the mitotic index 2.2-3.8% for normal epidermis, 4.2% for clinically un-

affected psoriatic skin and 21-22.7% for clinically affected skin [15]. Very recently, we have succeeded in purifying a novel, 45 kDa messenger-independent growth-related Ser/Thr protein kinase, named CK III [9, 10] which has features typical of the casein kinase family (especially CK II), but is clearly distinguished from protein kinases previously described in the literature. CK III takes part in the molecular mechanisms of normal and malignant proliferation of many organs such as the mammary gland, bone marrow, liver and urinary tract [8-10]. C K I I I has 3.4 times higher activity in psoriatic plaque epidermis than in unaffected tissue [11]. CK III, as well as CK II [13], is activated in response to insulin [8]. There is considerable evidence for the role growth factors in psoriasis [12]. CK II, which is present in keratinocytes [3], is significantly stimulated in response to several mitogens, including insulin, EGF and IGF-I [6, 7, 13]. From the above discussion, the high CK II activity in affectd psoiriatic epidermis is to be expected. It is possible that, CK II as well as, CK III [11] and other tyrosine [2] and Ser/Thr protein kinases [1], growth factors [12] are involved in the pathogenesis of psoriasis. Acknowledgements. We thank Dr. D. Petitclerc and Dr. J. Attal for helpful discussions, critical reading of the manuscript and stylistic advice.

113

References 1. Edelman A, Blumental D, Krebs E (1987) Protein serine/threonine kinases. Ann Rev Biochem 56:567-613 2. Gentleman S, Martensen T, Digiovanna J, Chader G (1984) Protein tyrosine kinase and protein phosphotyrosine phosphatase in normal and psoriatic skin. Biochim Biophys Acta 798:53-59 3. Hashida T, Yasumoto S (1990) Casein kinase II activities related to hyperphosphorylation of human papillomavirus type 16-E7 oncoprotein in epidermal keratinocytes. Biochem Biophys Res Commun 172:958-964 4. Hathaway G, Traugh J (1982) Casein kinases-multipotential protein kinases. Curr Top Cell Regul 21:101 127 5. Kiistala U, Mustakallio K (1968) Dermoepidermal separation with suction. Electron microscopic and histochemical study of initial events of blistering on human skin. J Invest Dermatol 48 : 466-477 6. Krebs E, Eisenman R, Kuenzel E, Litchfield D, Lozeman F, Liischer B, Sommercorn J (1988) Casein kinase II as a potentially important enzyme concerned with signal transduction. Cold Spring Harb Symp Quant Biol LIII: 77 84 7. Meek, D, Simon S, KikkawaU, EckardtW (1990) The p53 tumor suppressor protein is phosphorylated at serine 389 by casein kinase II. EMBO J 9:3253 3260

8. Mitev V, Sirakov L (1988) Insulin action in vivo on growthrelated protein kinase in lactating mouse mammary gland. Int J Biochem 20:97-100 9. Mitev V, Alexeev A, Angelov I, Marekov L. Purification of a novel Ser/Thr messenger-independent growth-related protein kinase from lactating goat mammary gland. C R Acad Bulg Sci (in press) 10. Mitev V, Angelov I, Marekov L. Identifcation and purification of a novel serine/threonine messenger-indepentent growthrelated protein kinase from lactating goat mammary gland. Biochim Biophys Acta (in press) 11. Miteva L, Mitev V, Dourmishev A. Casein kinase III in the pathogenesis of psoriasis. 18th World Congress of Dermatology, 12-18 June 1992, New York (in press) 12. Rothe M, Falanga V (I989) Growth factors. Arch Dermatol 125:1390-1398 13. Sommercorn J, Mulligan J, Lozeman F, Krebs E (1987)Activation of casein kinase II in response to insulin and to epidermal growth factor. Proc NatI Acad Sci USA 84:8834-8838 14. Tuazon P, Traugh A (1991) Casein kinase I and II - multipotential serine protein kinases: structure, function, and regulation. Adv Second Messenger Phosphoprotein Res 23:123-164 15. Weinstein G, McGullough J Cell proliferation kinetics (1991) In: Roenighk H, Mainbach H (eds) Psoriasis. Marcel Dekker, New York, pp 327-343