Phosphoinositide phosphorylation precedes growth in rat mammary tumors

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 134, No. 2, 1986

Pages 876-882

January 29, 1986

PHOSPHOINOSITIDE

PHOSPHORYLATION

Yoav Sharoni

PRECEDES GROWTH IN RAT MAMI~RY TUMORS

, Iris Teuerstein

and Joseph Levy

Endocrine Laboratory and Department of Clinical Biochemistry, Soroka University Hospital and Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Received December

16, 1985

DHBA-induced rat mammary tumors were used to study the possible association of phosphoinositide phosphorylation to tumor growth. These membranous enzymatic activities were measured during various stages of tumor growth induced by pharmacological manipulation of plasma prolactin level. An increase in phosphorylation of both phosphatidyl inositol and phosphatidyl inositol 4phosphate preceded the growth induced by prolactin concomitantly with an increase in tyrosine phosphorylation. Good correlation (r:0.87) existed between the tyrosine kinase activity and phosphatidyl inositol kinase activity of 21 individual tumors taken from animals at different stages of hormonal manipulation. Phosphoinositide phosphorylation was inhibited by quercetin and was not affected by cAMP, similar to tyrosine kinase. Phosphorylation of angiotensin II by tyrosine kinase was inhibited by 0.2 mg/ml phosphatidyl inositol 4 phosphate or phosphatidyl inositol 4,5-bisphosphate. ® 1986 Academic Press, Inc.

The studies

DMBA1-induced related

rat

to tumor

hormones.

Estrogens

tomy

antiestrogens

cause

(I),

growth

arrest

during prolactin protein

kinase

mammary growth,

tumor

and

or regression

preceded

tic activity was inhibited

which

the increase

i

(8) and the rat uterus

excellent

easily

(4).

for

manipulated

its growth, plasma

model

by

while ovariecprolactin

We recently

showed

level that

a 2-3 fold increase in membranal tyrosine in tumor area

(6), a bioflavonoid

been shown to inhibit the cAMP-independent mammary gland

an

is

decrease

of the tumors

by quercetin

as

rate

(2) stimulate

drugs

induced tumor growth,

activity,

serve

as its growth

(I) and prolactin (3)

may

(5).

This enzyma-

which had previously

protein kinase in mammary tumors

(9), as well as the protein kinase

(7),

(10) and

Author to whom correspondence should be addressed: Dr. Yoav Sharoni, Endocrinology L a b o r a t o r y , Soroka Medical C e n t e r , P.O. Box t51, Beer-Sheva 84101, Israel.

IAbbreviations used: DMBA - 7,12-Dimethylbenz(a)anthracene; PI - phosphatidyl inositol; PI 4-P - phosphatidyl inosltol 4 phosphate; PI 4,5-P 2 - phosphatidyl inositol 4,5-biphosphate; PKI - protein kinase inhibitor.

0006-291X/86 $1.50 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

876

Vo1.134, No. 2 , 1 9 8 6

phospholipid kinase-C

kinase

(12).

inositol

It

activities was

specific

(13).

of several

initiating

to

and

phosphorylation

increase

increase

for

suggested affected the

studies

receptor

in opposition

proliferation stimulation size

inositides

and

(5),

some growth

two

the

breakdown

the

activity is

protein with

of

second (15).

and

that operate

phosphorylation

polyoma

virus

to this hypothesis

phosphoinositide

middle

have shown (19,20).

kinases

have

with,

in a consecutive

tyrosine

kinase

activity

was

in this

report

we checked

shown

whether

is also related to hormone-induced

is thus

It was

or might

products

T-antigen

(18).

Recent

the possibility

synergistic

effect

order still exists. to precede

the

phospborylation

be

(11,16),

that the two activities

However, a

and

this pathway.

is associated

-

which

diacylglycerol

through

by

messengers

polyphosphoinositides

phosphates

(14).

Phosphoinositide

kinase activity of certain oncogene and

like PDGF

phosphoinositides

intracellular

amount

of processes

(17)

of

associated

factors of

trisphosphate

inositol

or that they operate of

and

proliferation

the

in the same protein molecule

tyrosine

tumor

to

by, the tyrosine

not reside the

the

that phosphoinositide

insulin

including

inositol

breakdown

the efficiency

cell

probably

1,2-diacylglycerol

available

pp60 v-src

Activation of this mechanism occurs in the signal

is

phospholipase-C

of

that

ligands,

reaction

can

(11)

suggested

lipid turnover

transduction The

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on

do

that cell

Just as

increase

in

of phospho-

mammary tumor growth.

MATERIALS AND METHODS Materials and methods. [~-32P]ATP was obtained from the Nuclear Research Center (Negev, Israel); DMBA9 quercetin, cAMP, theophylline, angiotensin II, PI, PI 4-P and PI 4,5-P 2 from Sigma Chemicals (St. Louis, MO); perphenazine from Schering Corp. (Kanilword, NJ); bromocriptine was a generous gift from Dr. E. Fluckiger, Sandoz Ltd. (Basel, Switzerland) Induction of tumors and preparation of membranes. Rat mammary tumors were induced by DMBA and their area was measured (7). Tumor growth arrest was achieved by daily injection of 0.5mg bromocriptine for 10-14 days. Rapid tumor growth was then induced by daily injection of 1.0mg perphenazine which causes an elevation of the plasma prolactin levels. The tumors were removed, frozen and crude membrane fractions were prepared as described previously (8). The membranes were dispersed in the homogenization buffer and were used for protein kinase assays. Tyrosine kinase activity. Angiotensin II was used as the peptide substrate as described previously (5,6). Incorporation of radioactivity into phosphopeptides was analysed both by absorption on phosphocellulose paper and by paper electrophoresis at pH 3.5.

877

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Phosphorylation of phosphoinositides. Tumor membranes were ~ c u b a t e d at 30°C in 30 1 containing 50mM Mes buffer, pH 6.5, 0.1mM [~-JLP]ATP (2-4 cpm/fmol). Phosphorylation of PI was measured with endogenous substrate in the presence of 20mM MgCI2, 0.1% triton X-IO0 for I min. Phosphorylation of PI 4-P was measured with 0.2 mg/ml exogenous substrate (sonicated for 2 min. at 45 °C) in the presence of 20mM MgC12, 10mM MnC12 and 0.1% CHAPS. The incubation time was 3 min. The reaction was initiated by the addition of a mixture of ATP with the divalent cations and terminated by the addition of 30 1 of 25mM ATP and 5mM sodium phosphate (pH 7.5) immediately followed by extraction in acid chloroform methanol (21). The various phospholipids in the extracts were separated by TLC on oxalate-impregnated silica gel plates with chloroform:methanol:4N NH40H (9:7:2) as the solvent. After autoradiography the appropriate spots w e r e excised and counted in a scintillation spectrometer.

RESULTS The changes in tumor area were observed during three phases of growth. first

phase

treatment

of

which

about

ten days was

showed

an average

a period

o£

relative

spontaneous

increase

growth

The

without

any

in tumor area of 2.0.

In

the second phase the rats were treated daily with bromocriptine for ten to fourteen

days.

continued shown).

to

During

the

grow

and

In the

third

with perphenazine. increase

(by

one

treatment

(Fig I).

first then phase

few

days

stopped

of

this

growing

the rats were

or

treatment even

treated

most

regressed

daily

for one

of

the

tumors

slightly

(not

to five

days

During this phase the tumors grew rapidly and a significant way

ANOVA)

in

tumor

area

was

evident

on

the

fourth

day

An almost fourfold increase in tumor area was evident on the

fifth day of treatment.

200 - ~ 60

|

4

<

C=

~

loc

T=

30

3

o

joE

2

E

Figure,

1:

of

0-E

0

0 1 2 3 4 5 Perphenazinetreatment (days)

1

Stimulation o f tumor g r o w t h and p h o s p h o i n o s i t i d e during perphenazine treatment. Tumor area at the end treatment (day O) was defined as one (relative area) during the next days of perphenazine treatment. phosphorylation was measured as described in the The results are the Mean + SEM of 4-8 tumors. denotes significant difference from day 0 (p
878

phosphorylation of bromocriptine and was followeO Phosphoinositide Methods section. by one way ANOVA

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS "B

250

E y=22+3X '~o-E! 15(I nr==0.87 / -l,m ' C- 200 21

[

o

E

o

~o

O ('

DAYSAFTERPERPHENAZINE ~REATMt.T , - 0 DAYS

10( "o ~

~o ~.

50

:8 7

/°o

o-3

....

•

~-5

....

•1

I 80 Angiotensin1IphosphorylQtion pmol.mg-Lrnin-I

'E'

20

Figure 2:

On

the

first

day

increase

added

4-P

that

in

reported

correlation individual

of

perphenazine

in the

the

I

60

by

us

membranes

previously tyrosine

for

there

was

of endogenous

(Fig.

kinase

tumors at different treatment)

treatment

phosphorylation

tumor

between

perphenazine

I

40

Correlation between PI-kinase activity and tyrosine kinase activity in tumor membranes. PI-kinase was measured by the phosphorylation of endogenous substrate. Angiotensin II (2mM) was used as the substrate for tyrosine kinase. The different symbols denote different days of perpbenazine treatment as marked in the figure.

threefold PI

....

o-1

I

I).

This

tyrosine activity

PI and of

increase

kinase and

already

(5).

We

PI-kinase

stages of the growth process

(Fig. 2).

was

a two-

to

exogenously parallel

analysed

activity

to the

in

(day 0 to day

21

5 of

The correlation coefficient was 0.87, streng-

thening the likelihood of an interrelationship

between the two types of kinase

activities. We

checked

phosphorylation

the

effects

(Fig.

3).

The

inhibited

by I0-4M quercetin,

the

stable

heat

shown).

The

inhibition

dose dependent, inhibition An

inhibitor

quercetin

phosphorylation

but was affected

of

and

the

cAMP

of

PI

neither

cAMP-dependent

of PI 4-P phosphorylation

achieved

4-P

phosphoinositide to

PI

4,5-P 2

was

by cAMP (Fig. 3a) nor by kinase

by quercetin (Fig 3b).

(results

not

(not shown) was The maximal

at 3.10-4M quercetin with both lipid substrates was 30-50%. question

are somehow

of these activities

on

protein

similar to that of PI phosphorylation

interesting

activities

of

was

related.

will

whether

tyrosine

Thus it is possible

interfere

and

phosphoinositide

kinase

that the substrate

for one

with the measurement

879

of the other

activity.

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

b 100'

o

P~P [

Ov 50

PIP2

i

ORIGIN. O

~ ~

~

o

~

0 ~I

0

Z

J

I

i

3_10-6 10-5 3_10-5 10-4 3_10-4

~

Quercetin [M]

C~ +

Fil~ure

Table

3:

I shows

substrate 0.2mg/ml

for

Effects of quercetin and cAMP on phosphoinositide phosphorylation in tumor membranes, a. Autoradiogram of a TLC showing the separation of lipids phosphorylated by tumor membranes in the presence of 0.2mg/ml exogenous PI 4-P. The radioactive PI 4-P is the product of endogenous PI phosphorylation, cAMP and quercetin were used at the indicated concentration (one representative experiment out of three), b. Effect of different quercetin concentrations on PI-kinase, measured with endogenous substrate. The results are the Mean + SEM of three experiments.

that

the

tyrosine

PI 4,5-P 2.

phosphorylation kinase,

At these

is

of

angiotensin

inhibited

concentrations,

by

0.2

II, which or

PI did not

is used

0.4mg/ml inhibit

PI

as the

4-P

or

tyrosine

by

kinase

activity. DISCUSSION The

discovery

associated

with

that

some

inositol

oncogene lipid

protein

turnover

products

(15)

and

stimulates

growth studies

factors of

several

TABLE I EFFECT OF PHOSPMOINOSITIDES ON TYROSINE KINASE ACTIVITY IN TUMOR MEMBRANE Phospboinosltide

Final Conc. pg/ml

n

NO additions

-

8

100

PI

200 400

11 3

96 + 14 121 + 23

PI 4-P

200 q00

11 3

35 + 8 30 + 21

PI 4,5-P 2

200

6

55 Z 16

32p Incorp. into angiotensin Percent ~ SEM

Tyroslne kinase was measured as described in the methods section. 'rumor membranes from different stages of perphenazine treatment were used. Activity without inhibition was between 22 and 97 pmol/min/mg protein.

880

are

Vol. 134, No. 2,. 1986

aspects

of

breakdown which

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphoinositide

in turn

activation

activate

protein

stores

tumor

activity

preceded

also

relationship and

exists tumor

observations:

these

As our previous

results

growth;

This

two

induced

by

the two enzymatic

we

now

enzymatic

conclusion

enzymatic

is

activities

perphenazine

activities,

was

phosphorylation

PI-kinase

kinases, 4,5-P 2

c.

of

by

these

this inhibition.

with

are

somehow

The inverse

inhibition

study provides

phoinositides

or

this

by tumor

by prolactin. activity

processes other

supported

by

the

following

simultaneously

a high activity

degree

during

of

may

membranes

of

may

PI.

clear,

and

was affected

found

indicate

reported

for

tyrosine

that

and

by 0.2mg/ml Although

the

to tyrosine PI

4-P or PI

physiological

they

suggest

that

some

specificity

the

be

involved

in

exists

in

kinase activity

by

the growth

(5).

affected

as it is

stimulation

directly

881

or

process

induced

of phosin these

with similar changes in tyrosine

Thus,

which operate through the PI cycle. activities

that the phosphorylation

correlates

the

two

cycle (24).

evidence

previously

by cAMP

that only part of

to growth

of the phospholipid

precedes

This increase

the

correlation

II (the substrate used for tyrosine kinase) was not checked

In summary,

activities

as between

inhibited

yet

related

the phosphoinositide

kinase

as well

related

was

not

known to stimulate

tumors

activities

inhibition

inhibition

processes

is

functional

(6), and in the lipid kinase activity

concentrations

results

that

kinase

The inhibition of phosphoinositide

70-90%

activity

tyrosine

of PI

a

b. Neither

the

lesser

kinase

similar

activities

angiotensin

This

is associated

not

significance enzymatic

(11).

Tyrosine

but

than

(22),

of Ca +2 ions

the pool

showed,

suggest

with

in mammary tumor membranes

of the pp60 v-src the

lower

phosphates

increases

change

or PKI, but both were inhibited by quercetin. phosphorylation

phosphoinositide

In this study we showed that which

these

the

and inositol

phosphorylation

tumor

between

stimulation

between

growth.

of

(23) and the mobilization

(22), respectively.

growth.

a.

result

glycerol

kinase-C

of phosphoinositide

4,5-P 2 precedes

growth

The

is the formation of 1,2-diacyl

from intracellular

them

cycle.

either of

one

or

both

of

the

growth

by

potentiating

This may involve protein kinase C indirectly

by

the

intracellular

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

messengers produced by the breakdown of polyphosphoinositides. may

operate

independently

of

protein

kinase

C

by

different

Alternatively it mechanisms,

as

suggested recently (25,26) or by some other unknown mechanisms.

ACKNOWLEDGHENTS This work was supported in part by grants from the Chief Scientist's Office, Israel Ministry of Health, from Israel Cancer Association and by the Beatty Malkin Grant for Cancer Research. The authors wish to thank Mrs. M. Duman for her excellent editorial assistance. REFERENCES I. Levy, J., Liel, Y. and Glick, S.M. (1981) Isr. J. Med. Sci. 17, 970-975. 2. Johnson, M.L., Levy, J. and Rosen, J.M. (1983) Cancer Res. 4_33, 2199-2209. 3. Levy, J., Liel, Y., Feldman, B., Aflallo, L. and Glick, S.M. (1981) Eur. J. Cancer 17, 1023-1026. q. Levy, J., Liel, Y., Feldman, B. and Sharoni, Y. (1983) Program of the Second International Congress on Hormone and Cancer, Monte Carlo 1983. J. Steroid Biochem. p. 43-S (Abstract). 5. Sharoni, Y., Radian, S. and Levy, J. (1985) FEBS Lett. 189, 133-136. 6. Levy, J., Teuerstein, I., Marbach, M., Radian, S. and Sharoni, Y. (1984) Biochem. Biophys. Res. Commun. 123, 1227-1233. 7. Sharoni, Y., Graziani, Y., Karny, N., Feldman, B. and Levy, J. (1984) Eur. J. Cancer Clin. Oneol. 20, 277-281. 8. Sharoni, Y., Feldman, B., Teuerstein, I. and Levy, J. (1984) Endocrinology 115, 1918-1924. 9. Sharoni, Y., Teuerstein, I., Shirman, A., Feldman, B. and Levy, J. (1984) Endocrinology 115, 2297-2302. 10. Graziani, Y., Erikson, E. and Erikson, R.L. (1983) Eur. J. Biochem. 135, 583-589. 11. Sugimoto, Y., Whitman, M., Cantely, L.C. and Erikson, R.L. (1984) Proc. Natl. Acad. Sci. (USA) 81, 2117-2122. 12. Gschwendt, M., Horn, F., Kittstein, W. and Marks, F. (1983) Biochem. Biophys. Res. Commun. 117, 444-447. 13. Michell, R.H. (1982) Cell Calcium 3, 429-440. 14. Doolittle, R.F., Hunkapiller, M.W., Hood, L.E., Devary, S.G.,Robins, K.C., Aaronson, S.A., Antioniades, H.N. (1983) Science 221, 275-276. 15. Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 315-321. 16. Macara, I.G., Marinetti, G.V. and Balduzzi, P.C. (1984) Proc. Natl. Acad. Sci. (USA) 81, 2728-2732. 17. Machicao, E. and Wieland, O.H. (1984) FEBS Lett. 175, 113-116. 18. Whitman, M., Kaplan, D.R., Schaffhausen, B., Cantley, L. and Roberts, T.M. (1985) Nature 315, 239-242. 19. Thompson, D.N., Cochet, C., Chambaz, E.M. and Gill, G.N. (1985) J. Biol. Chem. 260, 8824-8830. 20. MacDonald, M.L., Kuenzel, E.A., Glomset, J.A. and Krebs, E.G. (1985) Proc. Nat. Aead. Sci. (USA) 82, 3993-3997. 21. Oron, Y., Sharoni, Y., Lefkowitch, H. and Selinger, Z. (1978) in Wells, W.W. and Eisenberg, F. (eds.), Cyclitols and Phosphoinositides, pp. 383-397, Academic Press, New York. 22. Berridge, M.J. (1984) Biochem. J. 220, 345-360. 23. Nishizuka, Y. (1984) Nature 308, 693-698. 24. Smith, J.B., Smith, L., Brown, E.R., Barnes, D., Sabir, M.A., Davis, J.S. and Farese, R.V. (1984) Proc. Natl. Acad. Sci. (USA) 81, 7812-7816. 25. Lassing, I. and Lindberg, U. (1985) Nature 314, 472-474. 26. Burn, P., Rotman, A., Meyer, R.K. and Burger, M.M. (1985) Nature 314, 469-472. 882