Membrane phosphatase activity in somatic cell hybrids

Vol. 68, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MEMBRANE PHOSPHATASE ACTIVITY

IN SOMATIC CELL HYBRIDS

Michael J. Tisdale and Barry J. Phillips Department of Biochemistry, St. Thomas's Hospital Medical School, London SE1 7EH; and Chester Beatty Research Institute, Fulham Road, London SW3 6JB.

Received

October

21,197s

SUMMARY The dephosphorylation of membrane proteins by an endogenous phosphatase has been studied both in A9 and TLX5 cells and in hybrids between them. these cells differ both in growth rate and saturation density achieved --in vitro. The activity of the phosphatase seems to parallel the growth rate of these cell lines. It is considered that this phosphatase is part of an ATPase enzyme system. The enzyme from TLX5 cells is stimulated by cyclic adenosine 3'5'Prostaglandin El monophosphate (CAMP) and inhibited by zinc and fluoride ions. (PCEl) has been found to have no effect on the activity of the phosphatase. 'Ihe level by adenylate loss

cyclase,

by leakage

plasma

its

it

of breakdown medium.

is ideally

suited

led

The stimulatory

effects

to the suggestion

that

phosphorylated

Conversion direct

from

the

nucleophilic

by the action membrane

by its

rate

of production

by CAMP phosphodiesterase

As adenylate

to receive

cyclase

the stimuli

is

and its located

in the

of density

of cell

of membrane

cyclase

described

of these

phosphatase

exists

in the

the possible

dependent

between cells

phosphatase them.

is

form

thought

either

to occur or fluoride

involvement

of phosphorylation

of adenylate

has been measured The relationship

(4). 35

of adenylate

during

inhibition. in the

cyclase, both

between

by

exists form

on contact

(2).

ion,

Thus a possibility phosphorylated

(1)

active

dephosphorylated

by a hormone (3).

cyclase

a less

by a phosphatase

and the activity

0 1976 by Academic Press, Inc. of reprorlrrcfion in any form reserved.

adenylate

in two forms,

inactive

and in the activation

phosphoprotein

form

of phosphate

and is activated

growth

and in hybrids

acteristics

the enzyme exists

to the active

of a neighbouring

growth

on mammalian

form and a more active

inactive

To investigate control

of fluoride

displacement

adenylate

logarithmic

Copyright All rights

is determined

of growth.

or inhibited

cells

rate

to the external

membrane

inhibition

have

of CAMP in a cell

in

the activity A9

and

the growth

cyclase

TLX5

char-

has been

or that

Vol. 68, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS Cell culture Cells were grown in Dulbecco's modified Eagle's medium containing 10% foetal calf serum. Cultures were gassed with 10% CO2 in air. Cell lines were routinely passaged in Falcon plastic flasks, but cells were grown in 9cm plastic dishes for experimental purposes. An inoculum of A9 cells was kindly supplied by Professor H. Harris. Lines of TLX5 lymphoma cells were established from CBA/lAC mice bearing routine passages of the tumour. Hybridization The techniques used to induce cell fusion and to isolate hybrids will be described in a forthcoming publication (5). ASH/TLX was a clone isolated from a hybrid colony which arose after fusing A9 cells with TLX5 cells. ASH/TLX(tumour) was derived from the fourth passage of a transplanted ASH/TLX tumour grown in immune-deprived mice. The karyotypic characterization of the parent and hybrid cells is to be published (5). Both types of hybrid cell possessed approximately the full chromosome complement of one A9 and one TLX5 cell. Membrane phosphatase assal Cells were allowed to grow to confluence in plastic Petri dishes and removed with a rubber policeman. Cells (O.lml) were suspended in lml of 4GmM Tris-HCl, pH 7.2, containing 1lOmM NaCl, 1mM 1mM CaC12, 0.2% glucose, 0.3mCi of[32Pj' inorganic phosphate (sp.act. M&12, 206mCi/mmole) and either with or without 1mM dibutyryl CAMP (db CAMP), and were incubated at 37O for lh. After incubation, the cells were washed twice with 40mM Tris-HCl, pH 7.2 containing 1lCmM NaCl, centrifuging (300 x g for 3 min) between each wash. All subsequent operations were carried out at 4O. The cells were washed once more with 0.34M sucrose, resuspended in the sucrose solution and homogenized in a Teflon-glass homogenizer. A membrane pellet was then sedimented by centrifugation at 600 x g for 3 min, after which it was resuspended and washed three times in 40mM Tris-HCl, pH 7.2 containing 1mM MgC12. For the determination of phosphatase activity, membrane fragments (300-4OOpg of protein/ml) were suspended in 40mM Tris-HCl, pH 7.2, containing 10nM MgC12 at 37'. At various time intervals aliquots (4OOpl) of the membrane suspension were removed, centrifuged (3000 x g) for 10 min and 2OOpl of the supernatant was counted in lOm1 of scintillation fluid (naphthalene, PPC and diMe POPOP, in ethanol, dioxan and toluene) in a liquid scintillation counter (Packard, Tricarb model 3375). Protein was estimated by the method of Lowry --et a1.(6) using bovine serum albumin as standard. RESULTS Growth

behaviour

lines

used are

(doubling lhe most

of cell

cell

shown

time

in Fig.

in vitro 1.

16 h) and reached

number

declined

of the cells

from cultures

lines

very

appeared

These

results

dependent

growth

exhausted

or toxic.

inhibition

TLX5 cells the highest

rapidly

after

to be dead after

at day 5 supported

density.

Growth

suggested

only

had

this about

TLX5 cells

and continued

36

for

the highest

the

dividing

density

rate

was achieved

of fresh

until

cell

(11,000/mm2>.

7 or 8 days.

were

four

growth

maximum density

one division

that

curves

and

Medium

TLX5 cells

unresponsive

removed at low

to density

the medium was

Vol. 68, No. 1, 1976

BIOCHEMICAL

II

0

1

2

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3

4

5

6

Ttme

Pig.

1.

Growth curves for TLXS, ASH/TLX( tumour) (&-a>.

7

8

9

10

(days)

C-x>;

A9,

(o--o);

ASH/TLX

(-1

and

Cells were seeded in a large number of Petri dishes, at a density of 100 cells/mm2. At daily intervals, a number of dishes from each series was trypsinised and the cells were counted. The data were used to construct growth curves. In addition, the medium was removed from a number of dishes soon after they had reached maximum cell density and tested for its ability to support the growth of freshly seeded cells.

In marked division

contrast,

(doubling

ASH/TLX),

times

and lower

extended

plateau

ASH/TLX(tumourf

All

in which

the cells

reached

a density

of 2,600

(1,200

cells/mm2).

cells/mm2), Medium

cycle

removed

of culture

cells

showed

ASH/TLX(tumour),

phase,

than A9 (2,200

complete

of 19h for

maximum densities.

higher

a second

A9 and the hybrid

three

from

cell

from

exhibited for

which higher

cultures

rates

of

A9 and 24h for

lines

viable

cells/mm2,

these

growth

21h for

remained

and considerably

lower

an

several

weeks.

was slightly than

ASH/TLX

at day 7 supported

100 cells/mm2

to maximum

density. Characterization

of 32P phosphate

of enzymatic cell in

types

digestions is shown

the presence

incorporated

on the phosphorylated in Table

of pronase,

1.

In all

than with

cases, trypsin

37

into

membranes

membrane

The effect

components

more[32P]phosphate or phospholipase

of the four is

C.

released In some

w aJ

18

ASH/TLX(twnour)

57

23

29

22

Trypsin

hydroxylamine

94

a9

9

95

a4

Hydroxylamine

15

16

19

14

Ethanol - ether

on 32 P incorporated

95

C

extraction

21

21

41 a4

4

Phospholipase

solvent

63

Pronase

and organic

1

6

2

2

1

HCl

membranes

Chloroformmethanol-

in cell

membranes, prepared as described under 'Methods' were incubated for 30 min at 37O in 40 mM 7.2, containing 10 mM MgC12 and 5 mM ZnSO4 with the indicated enzyme (1 mg per ml). With pronase C, 20 mM CaC12. Membranes were also mixture also contained 10 mM MgC12 and with phospholipase 37' for 30 min in 100 mM sodium acetate buffer (pH 5.6) containing 1 M hydroxylamine. The samples and the radioactivity in the aqueous phase was determined by liquid scintillation counting. lipids was carried out with 1.4 ml mixtures of ethanol-ether (1:l) and chloroform-methanol-HCl and the radioactivity in the organic phase was measured.

22

ASH/TLX

Phosphorylated Tris-HCl, pH the incubation incubated at were centrifuged Extraction of (2O:lO: 0.1)

ia

A9

Control (no treatment)

15

Type

of enzyme digestions,

TLX5

Cell

Effect

Table

BIOCHEMICAL

Vol. 68, No. 1, 1976

le~.s[~~ P1.is

cases

in untreated

released

phospholipids

labelled

phosphate

Ihis

indicates

labile

acyl

is

that

mined

Membrane

proportional shows

dodecyl

the results

the total

membrane

Table

2.

Cell

Type

Under

membrane

proteins

cell

solvents

ethanol

-

(2O:lO:O.l)

does not

remove

sig-

of the extracted

product

polyacrylamide migrated

gel

was deter-

the conditions

electrophoresis

described,

by membrane protein

of

P1from 132

cells

incubated

and liberation membranes

remained

to the cathode. the rate

phosphatase

inorganic with

Table

phosphate or without

of radioactive

labelled

with

inorganic

32P

None +

0.90 1.11

Sk 6+

ASH/TLX

None f

0.84 0.84

3f 0.2 3 i= 0.2

ASH/l'LX (turnour)

None f

2.11 1.86

18 k 1.5 912

TLX5

None + to incubation of 1 mM db CAMP

with

32P inorganic

39

into

32P release 2 SEM pmole/mg protein/ min x 10'

6.4 10.4

0.5 0.5

28 -c 2 72 5 5 phosphate

2

db CAMP,

A9

* + refers

of

was

concentration.

32 P incorporation pmole/mg protein/h

Treatment*

through

the organic

and to membrane

of whole

from

1M hydroxylamine.

with

substrates

Rate of incorporation phosphate

100% of the

esters.

- HCl

of the incorporation fraction

with

may require

phosphoryl

product

of endogenous time

with

observed

through

sulphate

activity

to elapsed

almost

is

(7) when 9% of th e radioactivity

phosphorylated

phosphatase

cases

associated

lhe nature

t.1.c.

Sodium

dephosphorylation

is

C than

the phosphatase

the membranes

and not

of[32P.)-

the major

In all

- methanol

by two dimensional

on the origin.

from

the phosphate

or chloroform

that

action.

of the membranes

quantities

showed

its

bonds

of phospholipase

may indicate

released

phosphate

(1:l)

nificant

This for

Extraction ether

in the presence

membranes.

integral

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the presence

Vol. 68, No. 1, 1976

BIOCHEMICAL

and the unstimulated the four caused

cell

phosphatase

lines.

stimulation

into

activity

The addition

a 63% stimulation

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of isolated

membrane

fragments

of 1mM db CAMP to the incubation

of[32 P1. incorporation

A9 and a 12% stimulation

into into

medium

TLX5 membranes,

ASH/TLX(tumour).

of

a 22% The incor-

poration When the four of incorporation phosphate cells

were

of phosphate

into

the order

ranked

in order

a slight

had no effect stimulation

membranes,

stimulation

produced

3.

in

a 25% inhibition

the rate

shown in

of

when the

of release of various

Table

3.

neither

did

it

of L32P] ions

lhus, it

at a concentration

tested,

rate given

ZnS04 inhibited

of the enzyme,

rate

rate.

'Ihe effects

is also

by calcium

of increasing

or of increasing

growth

in Table

at the concentration

in order

was the same as that

an 80% increase

phosphatase

and NaF caused

pletely,

ranked

of increasing

as shown

of membrane

were

obtained

AMP causes

TLX5 membranes

activity caused

types

release,

Cyclic from

cell

on the

while

CaC12

almost

com-

of 5mM.

PGEl

reverse

the slight

ions. DISCUSSION

Membrane C3" PI phosphate the major

Table

by the intact

portion

3.

phosphatase

cells.

Effect

of various

from

TLX5 lymphoma

was measured

Enzymatic

of the phosphate

Agent Cyclic

activity

on the activity

Concentration AMP

CaC12 PGEl CaC12 + PGEl

digestion

was incorporated

agents

after

incorporation

studies into

showed

membrane

of membrane

Relative

179

1 mM

113

5 PM lmM+SnM

117

100

ZnSOd

1mM

19

NaF

5mM

75

40

that

proteins,

phosphatase

Activity*

1mM

of

Vol. 68, No. 1, 1976

either

BIOCHEMICAL

in the presence

90% of the labelled phosphate

is

phosphate

incorporated

Protein-bound in an ATPase

phosphates

reaction

(8).

transformed

cell

transformed

cells

with

(21h)

)

(9).

ASH/TLX

However,

activity

and that growth

blood

observed caused

(2).

(11)

been shown This

with

time

in both rate

16h)

ATPase

the normal

non-

normal

(10).

a decrease

and

'Ihe

have a high

endo-

in phosphatase

AS/TLX(tumour)

(19h)

result

is activated

>

by fluoride

of polymorphonuclear preparations activity

A9

PGEl.

enzyme,

phosphatases

with

TLX5 membrane

stimulation in

of the

Fluoride is

are

to

cortex

inhibited

by

by Zn2+ CAMP has

phosphatase.

activity

ion

similar

cerebral

phosphatases

and

lymphoma we have

which

from

and PGEl

granulocytes

from TLX5 with

of this

physiologically

lines

to be an inverse

line, basal

relationship

group.

of TLX5 phosphatase.

the regulation

of the phosphorylation

cyclase.

phosphatase

lowest

which

of phosphoprotein

a two-fold

For the cell

cell

that

of protein

our

may be important

appears

such

in the activity

A number

to cause

of adenylate

membrane

50% inhibition

in agreement

is

growth

the

as intermediates

with

of ATPase

with

there

of phosphatase

a 25% reduction

1OmM NaF (11).

compared

(doubling

of

of Na+-K+-activated

the levels

preparations

Using

no activation

the observed

rate

part

by many workers

activity

phosphatase

in membrane

platelets

phosphate

removed

(24h).

A phosphoprotein has been found

as an acyl

been correlated TLX5 cells

1M hydroxylamine probably

fibroblasts

show that

decreasing

Since

protein,

considered

greater

have also

phosphatase

activity

are

in transformed line

results

genous

from membrane

to five-fold

has been observed

of db CAMP.

in the membrane

acyl

A four-

present

or absence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

correlation

and the activity TLX5,

which

between

between

of the adenylate

shows

and fluoride

TLX.5, A9, ASH/TLX(tumour)

the highest

stimulated

the activity cyclase.

phosphatase

enzyme activity

enzyme dephosphorylation

41

and ASH/TLX

of the membrane 'l&us

activity (4).

and activity

there

the most malignant has both

Therefore, of the

the the

adenylate

Vol. 68, No. 1, 1976

cyclase

would

not

BIOCHEMICAL

appear

AND BIOPHYSICAL

to be indicated

M.J.T. wishes Acknowledgements receipt of a research grant.

RESEARCH COMMUNICATIONS

by the present

to thank

the Cancer

studies.

Research

Campaign

for

the

References Kornegay, D., Pennington, S. and Greenville, N.C. (1973) Fluoride 2,19-32. Najjar, V.A. and Constantopoulos, A. (1973) Molecular and Cellular Biochemistry 2,87-93. A., Judge, J.F.X., Rauner, R. and Najjar, V.A. 3. Layne, P., Constantopoulos, (1973) Biochem.Biophys.Res.Commun. s,800-805. B.J. (1975) Exptl.Cell Res. in press. 4. Tisdale, M.J. and Phillips, B.J. in preparation. 5. Phillips, W. J., Farr, A.L. and Randall, R.J. (1951) 6. Lowry, O.H., Rosenbrough, J.Biol.Chem.z,265-275. G., Fleischer, S. and Yamamoto, A. (1970) Lipids 5, 494-496. 7. Rouser, 2, 3275-3425. 8. Hokin, L.E. (1969) J.Gen.Physiol. L.B. and Friedman, H. (1974) Cancer Res. 34, 1862-1865. 9. Kasarov, 10. Elligsen, J.D., 'Ibompson, J.E., Frey, H.E. and Kruuv, J. (1974) Exptl. Cell Res, 87, 233-240. 11. Maeno, H. and Greengard, P. (1972) J.Biol.Chem. 247, 3269-3277. 1. 2.

42