Vol. 172, No. 2, 1990 October

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

30, 1990

Pages

958-964

CASEIN KINASE II ACTIVITIES RELATED TO HYPERPHOSPHORYLATION OF HUMAN PAPILLOMAVIRUS TYPE 16-E7 ONCOPROTEIN IN EPIDERMAL KERATINOCYTES Takashi

Hashida

Laboratory

SUMMARY:

The present

phosphorylated capable

The

kinase activity. levels

origins

fibroblasts.

These

differential

differential

transforminmg

E6 and E7 (l-3). transfected

in epidermal serine

CK II activities

activity

The E7 stimulates

are implicated

with those of Adenovirus

being

phosphorylated

* To whom correspondence 0006-291X/90 Copyright All rights

of E7 protein

The E7 protein by casein

contains kinase

The

also

play

958

oncogenes,

active E7 gene

E7 oncoprotein

binds

it’s biological

large T (5).

transforming interaction

to

functions

All these viral

activity of the E7 gene with RB (9), post-

a part

and serine-32

(CK) II (12).

$1.50

0 1990 by Academic Press. Inc. oj- reproduction in arly form reserved.

when biologically

and SV40

serine-31

should be addressed.

high

in human

two putative

(RB) and, therefore,

oncoproteins are phosphoproteins (6-8). Although appears to be achieved by it’s protein-protein phosphorylation

at significantly

of the activity

virus encoding

DNA synthesis

Ela

cell

for the casein

I ,93 AcldomlL ‘rr ss, Inc.

host cell (4).

gene product

from various

in host cells may play a part in the

type 16 is an oncogenic

susceptibility

translational

Extracts examined

levels

by E7-oncoprotein.

into an appropriate

but little in

(Ser-Ser-Glu-Glu-Glu)

that CK II activity was present

retinoblastoma

activity (g-11).

residues

by HPV 16 were

of

We found that highly

keratinocytes

by casein kinase II (CK II). transformed

of E7-oncoprotein

keratinocytes.

but little or no detectable

Human papillomavirus was

contains

The results showed

in keratinocytes

241, Japan

hyper-phosphorylation

was present

E7 oncoprotein

human

Institute

Yokohama

study describes

of being phosphorylated

lines including

Asahi-ku,

(HPV) type 16 in epidermal

E7-oncoprotein

fibroblasts.

and Cell Biology

1990

20,

human papillomavirus

Yasumoto*

Cancer Center Research

54-2 Nakao-cho,

September

Shigeru

of Molecular

Kanagawa

Received

and

in their doublets

transforming capable

of

The CK II activity has been

Vol.

BIOCHEMICAL

172, No. 2, 1990

implicated

in processes

of cell growth,

AND BIOPHYSICAL

though

RESEARCH COMMUNICATIONS

it’s biological

significance

is poorly

CK II activities

in different

cell

documented. To study harboring

HPV 16 genomes

oncoprotein fibroblasts. differences provide

epitheliotropic

HPV functions, were

The

level

evidence

between

that epitheliotropic

CK II activity in epidermal

in epidermal

of the differential

of CK II activities

In this study we found that HPV 16 E7

examined.

was highly phosphorylated

lines

keratinocytes

E7-phosphorylation keratinocytes

virus functions

comparing was

comparable

and fibroblasts. partly reflect

to those in The

in significantly

with results high

keratinocytes.

MATERIALS

and METHODS

Cell culture: Primary epidermal keratinocytes were prepared from human foreskin epidermis or surgically dissected adult human skin regions and back skin epidermis of newborn BALB/c mouse by the method of collagenase-floating technique (13). The cells were maintained in the complete MCDB152 medium containing a hormone mixture: epidermal growth factor (EGF) (10 rig/ml), insulin (5 yglml), transferrin (10 pglml), hydrocortisone (0.2 FM), phosphoethanolamine (5 PM), ethanolamine (5 PM), and supplemented with 0.5% chelex-treated fetal calf serum (cFCS) and bovine The culture medium was replaced pituitary extract (BPE, 50 pg proteins per ml). every other day and the cells with 70% - 80% confluence were subcultured at a ratio of 1 : 5 splits. Only growing keratinocytes can be maintained under these culture conditions, and fibroblast contaminants were negligible. Primary human and mouse fibroblasts were prepared from human foreskin dermis and back skin dermis of new born BALB/c mouse, respectively. These cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Construction of recombinanfs: PvulIIPstl fragment (nt.,551-875, 324 bp long) containing E7 ORF blunt-ended by Si nuclease, was cloned at blunt-ending BamHl recognition sites created by Sl-nuclease digestion of partially cleaved pcD2 vector DNA by BamHl. The recombinant was designated pDE7 (Fig. 1). Plasmid pMHPVl6d contains a head to tail dimer of HPV 16 DNA cloned at BamHl site of the vector pdMMTneo (14), which carries neomycin resistant gene. DNA transfection and the established cell lines: Viral DNAs were transfected into primary human keratinocytes by either Ca 2+-phosphate coprecipitation method (13) or electroporation using Baekon 2000 Advanced Gene Transfer system (Baekon, Five clonal Saratoga, CA). Ten to 15pg of DNA were used for 1 x 106 keratinocytes. human keratinocyte cell lines were established. These cell lines continued to grow beyond 300 population doubling. Mouse keratinocytes were transfected with DNA using the method of modified calcium phosphate precipitation (13), then selected by G418 (100 Jrg/ml) for 2 days. G418-resistant cells and cell lines were subjected to further investigation. Human and mouse fibroblasts were transfected by the standard calcium phosphate technique. Human fibroblasts transfected with pMHPV16d or pMHPV16s were selected by G418 (100 pg/ml). Unlike keratinocyte cell lines, two fibroblast lines designated PHFblGd and PHFblGs senesced with a limited life span. An averaged life span was 150 population doublings until they senesced. All these human cell lines harbored transcriptionally active HPV genomes (Fig. 2) and 1.8 kb mRNA is a major transcript containing E6/E7/El “E4/E5 (14, 15). Metabolic labeling and immunoprecipitation: exponentially growing cultures were labeled for 5 h with either 0.1 mCi/ml [35S] cystein or 0.1 mCi/ml [32P] orthophosphate per 90 mm dish. Cells was lysed using 0.5 ml of HSN buffer (250 mM NaCI, 0.1 % Nonidet P-40, 50 mM HEPES, pH 7.4, 1 mM phenylmethylsulfonyl fluoride [PMSF] and 1 pg/ml antipain). After 30 min on ice, cells were scraped off the plates and cell debris was removed by centrifugation at 10000 r.p.m. for 10 min at 4’C. Aliquots containing equal amounts of radioactive trichroroacetic acid-insoluble 959

Vol.

172,

No.

2, 1990

Bgll

Pvull 0

562

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Hindll

Pstl

2000

551 st5lOOCJ

L

BIOCHEMICAL

I

I

8551859

-----

2.4-

1170

1.4-

7

E6

sjyo4 1

01

----_

Elb Figure

1.

HPV 16-E7

gene

02

--w-m cloned

in pcD2

expression

vector

Figure 2. HPV 16-early gene mRNAs expressed in human cell lines. were hybridized with [32P] labeled EcoRIIPstl (nt. 7454-875, 1.3 kb DNA segment containing only E6/E7 reading frames.

Northern blots long) HPV 16

materials were incubated at 4°C with antiserum against HPVl6 E7 protein (16). Then, 50 ~1 of protein A-Sepharose (Pharmacia) in HSN buffer was added. After the incubation for 1 h at 4’C, the protein A-Sepharose were washed four times with 1 ml of HSN buffer at 4’C. The proteins were then analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography or autoradiography. Equivalent volumes of cell extracts from parallel cultures of cells labeled with [32P] orthophosphate were also immunoprecipitated. Casein Kinase Assays: Cell extracts were prepared as described elsewhere (17). Briefly,medium was removed and the plates were washed four times with 5 ml of chilled PBS. After the final rinse, the cells were harvested in 2 ml of extraction buffer (80 mM ,&-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, pH 7.3) and sonicated. The homogenates were sedimented at 150,000 x g for 60 min, and the supernatants were in the recovered and frozen in liquid nitrogen until use. Total protein concentrations extracts were determined by the Bio-Rad Coomassie Blue assay using bovine plasma gamma globulin as a standard. Aliquots of extracts were incubated in a final volume of 50 ~1 at 3O’C for 10 min in the presence of 20 mM HEPES, pH 7.4, 20 mM MgCI2, 100pM [f-32P]ATP (2000-5000 cpm/pmol) and with casein (5 mg/ml) or with no added substrate. Reactions were An aliquot of the reaction mixture was spotted onto started by adding radioactive ATP. Whatman P81 chromatography paper and the reaction was terminated by washing the papers with 10 mM H3P04 as described by Glass et a/.(18). Radioactivities of the dried filter papers were counted in Aquasol(NEN Res. Products) by using liquid Kinase activity was calculated on the basis of net phosphorylation scintillation counter. of added substrate. 960

Vol. 172, No. 2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS Differential

expression

study whether and

HPV i&E7

fibroblasts,

is differentially performed

significant

phosphorylated

Table

1.

more

efficiently

These

protein

kinase

(Table

m 1

35S

labeled

types.

cystein

or [32P]

Relatively

high

and PHFb 16d cells E7 protein

suggest

Similar

was only

experiments

(Fig. 3B).

Again,

phosphorylated

than

panel

analysis

that phosphorylation

were

The E7 oncoprotein

cell lines (Fig.SB,

by densitometric

only

detectable

serine residues kinase activities 2).

casein.

The

enzyme

consensus in various activities

right).

E7 The

and summalized

of E7 oncoprotein

in fibroblasts

reflecting

in keratinocytes:

in was

differential

Since

to be a substrate were

normalized

1234

by HPV 16 were

with the amount

6

7 32

6

9

P

32P

Figure 3. Phosphorylation of HPVlG E7 protein in transformed cell lines. (4 Human cell lines harboring full-length HPV 16 DNA or (B) mouse lines transfected with pDE7 DNA were incubated with (35Sjcystein or [32P] orthophosphate. The labeled cellular proteins were immunoprecipitated with anti-serum against HPVl6 E7 protein. SDS-PAGE were performed using 15% gel. (M), molecular weight markers in kilodaltons. 961

of

in all cell lines including

5

35S

5676

HPV 16-E7

of CK II (Ser-Glu-Glu-

cell lines transformed

The casein kinase activity was detected

/ 234

[35S]

in PHKl6d-I

to

in keratinocytes

to SDS-PAGE.

by the E7 gene alone.

in keratinocytes

II activity

Glu) (Fig. 4), casein

3.0

In order

activities.

contains

phosphorylated

cell

in keratinocyte

were quantified

results strongly

kinase

oncoprotein

either

of [32P]

from all transformants

achieved

protein:

The results show that HPV 16-E7 oncoprotein

in different

was only detectable

of phosphrylation

measured

line.

using mouse cell lines transformed

oncoprotein

with

E7

phosphorylated

and subjected amounts

PHKlGd-I

was immunoprecipitated

Casein

labeled

with 18.5 kd was detected

In contrast,

in keratinocyte

extents

proteins

of E7 protein

of

is differentially

were immunoprecipitated

(Fig. 3A). present

phosphorylation

oncoprotein

cellular

orthophosphate expression

and

Vol.

172, No. 2, 1990

Table

I.

BIOCHEMICAL

Phosphorylation

AND BIOPHYSICAL

of

E7

protein

(‘iP/‘5SmE7

Keratinocytes Human

cells

Mouse

cells

PMKH7-3

PMFP7-

I.

0.

0.

by

calculated

relative

inhibits

keratinocyte

casein

ratio

blocked.

in human

E7 protein

of relatively

I

PMFP7-2

E7

34>

Fig.Gcvert 01‘

films

X-ray

and

protein.

cell lines.

In the presence

of heparin

50% of the kinase activity in the human

In contrast,

fibroblasts.

no heparin

These

only detectable

results

in keratinocyte

heparin-sensitive

high concentration

08>

0. in

of

kinase II, approximately

we detected

0.

14>

bands

protein)

PHFhlGs

scanning

the activity varied among

activity was detected

presence

protein “P/35S

cell lines were

phosphorylated

81

densitometric

origin, though

fibroblasts,

00 labeled

quantified

which

00

PMKH7-2

Radioactively

fibroblast

Fihroblasts .____~ PHFhlGd 0. 03>

PHKlG-I I.

RESEARCH COMMUNICATIONS

CK activity of heparin

sensitive

casein

are comparable cell lines.

in mouse

kinase with the

Unlike

fibroblasts

(1.0 pg/ml)(Table

human in the

2).

DISCUSSION We have shown kinase

here that

activity than dermal

undetectable

in human

tha activity reflecting comparable

with extents kinases

fibroblasts.

fibroblasts

CK II activities

Casein

have been

*‘AsnAspSer

HPV18

E7

“‘Leu

Ser

HPVla

E7

3*Pro

SV40

LT

heparin

sentitive

concentrations

in mouse fibroblasts. epidermal

identified

processes

E7

pocess significantly

in a range of higher

of hyper-phosphorylation

in physiological

Ela

Notably,

between

HPV16

Ad5

keratinocytes

CK II was present

differential

implicated

epidermal

CK activity of heparin

was where

The results show

keratinocytes

and

fibroblasts

that were

of HPV 16-E7 oncoprotein.

in a variety

of protein

higher casein

of mammalian

phosphorylation

related

cells and been to cell growth

Ser

Glu

Glu

Glu

ASP

GIuq7

ASP

Ser

Glu

Glu

Glu

Asnu3’

ASP

ASP

Ile

Glu

Glu

Glu

Leu

Va140

‘*¶Phe

Pro

Pro

Ser

Asp

ASP

Glu

ASP

GIu’~~

“@Pro

Ser

Ser

Asp

Asp

Glu

Ala

Thr

Ala”’

Figure 4 Serine residues (bold) of the HPV 16. and 18-E7 proteins capable of being phosphorylated by casein kinase II. Underlines indicate acidic stretch of amino acid containing m and Bsp residues comparing with those of HPV la, Ad5 EIA and SV40 large T.

962

BIOCHEMICAL

Vol. 172, No. 2, 1990

Table

The

2.

difference

betneen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

heratinocrtes klnase

Total Gel

I

extracts

activity

fibroblasts

and

II

of

casein

inhibition

% protein)

by

case!"

kinase

Human

mInIme.

P r

ii

(as/ml)

0

n.:

I

.n

keratinocytes PHKIGd-I

48.2

27.9

24.3

50

23.9

YHKlfid-II

34.2

16.3

I 9

Ii

43

17.9

PHKIGL-I

7.1.

7.1

5

6

73

Ii.4

PHKl6L-2

13.9

8.2

5.8

Human

I

8.

58

I

fibroblasts 14.2

PHFbl6d PH

Fbl

I

14.0

NHF

Mouse

15.2

0

.c

0.7

I 7

0

IS.0

I

‘.

0.

I 3

6

15.3

0

q

0.4

13.5

15.

6s

I

keratinocytes PMKH7-2

Mouse

29.3

27.7

IO.8

64

15.7

16.3

11.7

26

19.

I

fibroblasts PMFP7-1

and differentiation

(19).

different cell types.

SV40

of heparin

activities

of various

large T, HPV16-E7

essential Serine

for their biological residues

phosphorylation

(20, 21).

viral

contains

based on sensitivity to an inhibitor This

enzyme

oncoproteins

(7-9).

acidic stretch

of amino

activities

in the acidic regions at Ser-31

4.0

CK, type I and type II, have been found in many

CK II can be distinguished

as low concentration biological

Two distinct

such as binding are a potential

and Ser-32

transforming

activity of this oncoprotein

transforming

activity in human

activity

in

Like adenovirus ElA acids whose functions

and are

to RB or transforming

CK II phosphorylation

of E7 by CK II appears (9, il),

fibroblasts

relatively

such

is implicated

activity.

site.

to be necessary weak

Since for full-

HPV 16-associated

may be partly due to the differential

CK II

activities. HPVs

are highly

carcinomas

epitheliotropic

virus

in natural

but rarely with fibrosarcoma.

transforming

activity

only

epithelial

using

unpublished

results).

the differential

regarding

These differential

transforming

activities

(22)

and are

associated

with

assay,

their

Using in vitro transformation

immortalization

keratinocytes

but

of natural not

dermal

CK II activities by HPV 16-E7

host can be demonstrated human

fibroblasts

(13,

our

in host ceils may play a part in oncopro!ein.

ACKNOWLEDGMENTS We wish to thank Dr. K. Yoshiike for the gift of anti-E7 technical

assistances.

Research

Programs

I"

activity

heparin CPRUI

Hepailn

case

Heparin-sensitive

kinnses

(pmol/mln/mg

in

activity

This work was supported from the Ministry of Education, 963

antiserum,

and H. Andoh

partly by Grants-in-Aids Science,

for

for Cancer

and Culture, Japan.

1 e

in

1

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNlCATfONS

REFERENCES 1. Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy, D. R., and Schiller, J. T. (1989) EMBO J., 8, 3905-3910. 2. Munger, K., Phelps, W. C., Bubb, V., Howley, P. M., and Schlegel, R. (1989) J. Virol., 63, 44174421. 3. Watanabe, S., Kanda, T., and Yoshiike, K. (1989) J. Virol., 63, 965969. 4. Sato, H., Furuno, A., and Yoshiike. (1989) Virology, 168, 195-199. 5. Dyson, N., Howley, P. M., Munger, K., Harlow, E. (1998) Science, 243, 934937. 6. Luther, L. A., Loewenstein, P. M., and Green, M. (1985) J. Viral., 56, 183-193. 7. Smotkin, D., and Wettstein, F. 0. (1987) J. Virol., 61, 1686-1689. 8. Paucha, E., Kalderon, D., Harvey, R. W., and Smith, A. E. (1986) J. Virol., 57, 50-64. 9. Barbosa, M. S., Edmonds, C., Fisher, C., Schiller, J. T., Lowy, D. R., Vousden, K. H. (1990) EMBO J., 9, 153-160. 10. Edmonds, C., and Vousden, K. H. (1989) J. Virol., 63, 2650-2656. 11. Watanabe, S., Kanda, T., Sato, H., Furuno, A., and Yoshiike, K. (1990) J. Virol., 64, 207-214. 12. Kuenzel, E. A., Mulligan, J. A., Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem., 262, 9136-9140. 13. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J., and DiPaolo, J. A. (1987) J. Virol., 61, 1061-1066. 14. Yasumoto, S., Doniger, J., and DiPaolo, J. A. (1987) Mol. Cell. Biol., 7, 21652172. 15. Taniguchi, A., and Yasumoto, S., (1990) Virus Genes, 3, 221-233. 16. Sato, H., Watanabe, S., Furuno, A., and Yoshiike, K. (1989) Virology, 170, 31 l315. 17. Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem., 262, 3839-3845. 18. Glass, 0. B., Masaracchia, R. A., Feramisco, J. R., and Kemp, B. E. (1978) Anal. Biochem. 87, 566-575. 19. Hathaway, G. M., and Traugh, J. A. (1982) Curr. Top. Cell. Regul., 21, 101. 127. 20. Maenpaa, P. H., (1977) Biochim. Biophys. Acta, 498, 294-305. 21. Hathaway, G. M., Lubben, T. H., and Traugh, J. A., (1980) J. Biol. Chem., 255, 8038-8041. (N. P. Salzman 22. zur Hausen, H., and Schneider, A . (1987) In The papovaviridae and P. M. Howtey, Eds.), Vol. 2, pp. 245-253. Plenum Publishing Corp., New York.

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