Carboxyl terminal tyrosine metabolism of alpha tubulin and changes in cell shape: Chinese hamster ovary cells

Vol. 100, No. 4,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1642-1650

June 30, 1981

CARBOXYL

TERMINAL

TYROSINE

IN CELL

Grace Ribicoff

G.

Research

METABOLISM

SHAPE:

Deanin,

CHINESE

Susan

Center,

Norwich,

University

Received

May 22,

OF ALPHA

of

HAMSTER

F.

Preston,

CT,

and

TUBULIN

OVARY

and

Malcolm

Department

of

Connecticut,

AND

CHANGES

CELLS

W.

Gordon

Biobehavioral

Storrs,

Sciences,

CT

1981 SUMMARY

Chinese hamster ovary cells maintained in their epithelial-like form not incorporate tyrosine post-translationally into c1 tubulin even though significant fraction of the soluble tubulin is tyrosinated and tubulin:tyrosine ligase is present. Incubation with dibutyryl cyclic AMP + testosterone, which leads to a change in cell shape, immediately activates this metabolic pathway . Podophyllotoxin, which prevents the conversion to a fibroblast1 i ke morphology, significantly inhibits this activation.

do a

INTRODUCTION There

is

metabolism

of

measures, tyrosinable

of

carboxyl

in

terminal change

Changes

in

cell

shape

are

generation

the of

ligase

the

total

tyrosine

this

terminal

cellular

(1,2),

(3,4)

pool metabolic

tyrosine

asymmetries.

activity

tubulin

(5),

carboxyl

By

the

and

pathway

the is

several

relative rate

amount of

turnover

stimulated

when

shape. membrane

morphology

carboxyl

by

we

as

well

cytoskeletal

terminal

reorganization,

dependent

the

accompanied

of

between

tubulin:tyrosine

to

skeletal

and

tubulin

stimulation

the

more

metabolism

considered

the

obvious

changes

(6,7).

reorganization

tyrosine

have

as

is

correlated

possibility

that

in

Since

the

with

cyto-

they

are

inter-

(8).

Dibutyryl cells.

c1 tubulin

begin

cell

correlation

including

of

cells

a positive

cyclic In

ABBREVIATIONS:

Sertoli

AMP cells,

CHO, TCA,

promotes these

changes changes

Chinese hamster trichloroacetic

in

presumably ovary; acid.

0006-291X/81/121642-09$01.00/0 Cop.vrighr G 1981 b-vAcademic Press, Inc. All rights of reproduction in any form reserved.

shape

1642

bu2cAMP,

in

a variety

involve dibutyryl

of

disruption cyclic

cultured of AMP;

bridges

BIOCHEMICAL

Vol. 100, No. 4,198l

between

microtubules

duces

a transient

shape

from

the

transformation” change

in

sence

of

are metabol lead

to

reverse

of

(10).

In

to

(11,12).

Reverse

membrane or

should

be

by

in

12 cells,

cells,

a change

that

CHO

cells

AND

METHODS

been

preceded does

(12). of

can

has

If

not

be

an

occur

in

by

“reverse immediate in

cytoskeletal

the

pre-

changes

terminal under

in-

induced

called

by

carboxyl incubated

bu2cAMP

tyrosine conditions

that

transformation.

CHO-Kl cells were grown Plastics) in 5 ml of Ham’s serum (Gibco), 85 units/ml moved after trypsinization Culture medium evaluation. studies

is

poisons

MATERIALS

For

that

a change

observed

COMMUNICATIONS

morphology

transformation

a stimulation

PC

CHO-Kl

a process

microfilament

RESEARCH

In

a fibroblast-like

morphology,

accompanied it

neurites

+ testosterone,

microtubule

ism,

(9).

bu2cAMP

plasma

necessarily

bundles

epithelial-like of

BIOPHYSICAL

microfilament

formation

an

addition

and

AND

of

incorporation

on 100 mm polystyrene tissue culture dishes (Falcon F 12 medium supplemented with 10% dialyzed fetal calf Cells were repenicillin and 30 ug/ml streptomycin. and replated 3-4 days prior to their experimental was changed daily except where noted. of

tyrosine,

cultures

were

pulsed

with

[3H]-

tyrosine ( 50 $i/plate) for the time periods designated, following which the radioactive medium was removed, the cells washed 5 times with phosphatebuffered saline and then collected by scraping with a rubber policeman into 5 ml of buffer containing 100 mM 2-(N-morpholino)ethane sulfonic acid, pH 6.6, 0.5 mM MgC12, 1 mM ethyleneglycol-bis-(B-aminoethylether)-N,N’-tetraacetic acid, 2 mM 2-mercaptoethanol, 0.5 mM GTP and 10% glycerol. The suspension was sonicated for 15 set at setting #2 of the Kontes micro-ultrasonic cell disrupter, the volume adjusted to 0.6 ml with additional buffer and centrifuged for 1 h at 100,000 x g at 2°C. Samples of the supernatant fraction were removed for the evaluation of protein (13), precipitation with 10% TCA for the determination of solubl radioactivity and tyrosine concentration (14) and for the incorporation of [ s HI-tyrosine into TCA-precipitable protein by the disk procedure ( 15) . The remaining supernatant was incubated with vinblastine sulfate under conditions that quantitatively precipitate tubulin (8,16). The tubulin was dissolved and fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis (17); radioactivity associated with ~1 and S tubulin was determined as described previously (5,8). Tubulin:tyrosine ligase activity in CHO cell extracts was determined after the addition of 3x cycled rat brain tubulin (18). The tubulin concentration in CHO supernatant fractions was determined by colchicine binding (19) and the distribution of tubulin into non-tyrosinable and tyrosinable species was measured as described by Nath and Flavin (20). [Ring-2,6-3H]-L-tyrosine [3H]-colchicine (19.6 tion. Testosterone, boxypeptidase A were chased from the Aldrich

(approximately 50 Ci/mmol) and [ring C, methoxyCi/mmol) were purchased from New England Nuclear Corporabu2cAMP, vinblastine sulfate, podophyllotoxin and carobtained from Sigma. The R-phenylpropionate was purChemical Co.

1643

Vol. 100, No. 4,1981

A.

TOTAL

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

6. NBULIN

PROTEIN

Fig. I. Translationa 11 Incorporation of Tyrosine into Protein in CHO Cells. Cel Is were incubated in fresh F 12 for I h before [3H]-tyrosine pulse (o-o-o); in fresh F 12 with 0. I5 mM bu2cAMP and 0.05 mt4 testosterone for 1 h (A-A-&); or in 24 h conditioned medium (o-o-o). Incorporation of tyrosine into total protein was measured at each of the designated time periods by the disk procedure (IS), and into tubulin after fractionation of vinblastine sulfate precipitates on polyacrylamide gels (see Methods). Incorporation of tyrosine was calculated on the basis of the specific activity of the cytoplasmic tyrosine pool.

RESULTS When like

CHO

morphology

tyrosine to

rapid;

of

the

medium

under

pulsed

incorporated

for at

incorporate

transport. is

cultured are

is

Failure

5 min,

cytoplasmic

pool

is

710

dpm/pmol.

the

for

to

the is

min

carboxyl

700

with

rapid

is medium time

linearity total

the

c1 chain

not

due

to

inhibition

and

the

cytoplasmic

measured,

and Nor

1644

that of tubulin

is

it

radioactive

of

tubulin. of

tyrosine

tyrosine the

incorporation

protein

equilibration.

while

epithelialno

of

tyrosine of

their

r3Hl-tyrosine,

period

dpm/pmol

both

maintain

terminus

the

earliest

into this

90

that

tyrosine

The

processes evidence

up

between

within

DISCUSSION

conditions

tritiated

Equilibration

translational tional

cells

AND

of labeled (Fig.

possible

pool

specific the

activity culture tyrosine

by

1)

is

addi-

to

explain

BIOCHEMICAL

vol. 100, No. 4,198l

1

1

5

I5

AND BIOPHYSICAL

RESEARCH

30

COMMUNICATIONS

I

h

45

SO

,

Time (minutes)

Fig. 2. Post-Translational Incorporation of Tyrosine into Tubuiin in CHO Ceils. Cells were incubated with 0.15 mfl bu2cAMP and 0.05 mM testosterone for 1 h (o-o-o) or 18 hr (o-e-e); or with 0.03 mM cycioheximide for 1 h (A-A-A). Incorporation of tyrosine at the carboxyi terminus of a tubuiin was measured after fractionation of vinbiastine sulfate precipitated tubuiin on poiyacryiamide gels and calculated as described in Fig. 1.

the

failure

either of

to

tubulin the

these

incorporate or

c1 chain. cells

supernatant

the

tyrosine enzyme

at

that

fairly

protein/

h,

the

ligase

high,

carboxyl

catalyzes

Tubulin:tyrosine is

the

1 nmol

tubulin

represents

by

the

post-translational

activity

approximately

while

terminus

in of

extracts tyrosine

about

absence

of

modification prepared

from

incorporated/mg

5% of

the

total

soluble

protein. Conditions boxy1 for

which

terminal as

CI carboxyl

little

induce

tyrosine as terminus

reverse

transformation

incorporation.

10 min (Fig.

results 2).

Treatment in

During

the

incorporation the

1645

60 min

immediately with

bu2cAMP of

incubation

activate and

labeled

cartestosterone

tyrosine period,

about

at

the

15

Vol. 100, No. 4,1981

BIOCHEMICAL

AND BIOPHYSICAL

IS

pmols

of

tyrosine

tubulin

now

are

contai

Cycloheximide,

which

formed

f ibroblasts

tyrosine

into

Carboxyl incubated min natant

and

ns

a tubuli terminal

with ceases protein

on Post-Translational Incorporation of Cells. Cells were incubated for 1 h with 0.15 in fresh F 12 medium + (A-A-4) and - (A-A-A) h conditioned medium + (o-o-o) and - (o-o-o) of tyrosine into a tubulin was measured as

supernatant

a newly

incorporated

i nduces

reverse

also

n in

after

the

(Fig.

2).

(Fig.

its

of

in

for

18-24 of

1s

4% of

carboxyl

hamster

ml4

the

term #Gnus. sarcoma

trans-

incorporation

of

2).

incorporation

ccl

almost

post-translational

incorporation These

at

transformation

activates

bu2cAMP-testosterone

protein;

tyrosine

CHO cells

tyrosine

80

(minutes)

incorporated/mg

(21 ),

COMMUNICATIONS

43

30 The

Fig. 3. Effect of Podophyllotoxin Tyrosine into o Tubulin in CHO bu2cAHP and 0.05 mM testosterone 5 UM podophyllotoxin; or in 24 podophyllotoxin. Incorporation described in Fig. 2.

RESEARCH

are

1646

CHO

cells

that

h proceeds

for

about

6 pmols

of

fully

reverse-transformed

have

been

approximately

15

tyrosine/mg

superand

have

8lOCHEMlCAL

Vol. 100, No. 4,198l

Table

1.

Culture

The

Distribution

of

AND BIOPHYSICAL

RESEARCH

Tubulin

into

Non-Tyrosinable

Several

Incubation

Condition

Tyrosinable

and

Tyrosinable

Control

30

Non-Tyrosinable

% of total tubul in

in

24

h medium tubulin t3H]-tyrosine

The tyrosinated without prior

tubulin treatment

The non-tyrosinable sample as measured as the average of

found cultured

for

“freshly

nor

stimulate

k

0.2

0.86

f

115.1

f

7.0

25.3

+ 1.5

4.9

2

0.3

1.1

k 0.07

f

5.4

20.0

fr 1.2

2.5

f

0.15

0.55

*

is

determined incorporated

no

is

as and

3).

cells

for

0.05

rat

inhibitor

% of total tubul In

340.3

74.9

334.5

73.6

361.3

79.5

is

addition

appears metabolism

by most

ligase not

of

more

than

(22).

modified

the total tubulin.

in

cycloheximide

any

from

these

at

of

cells

have in

been

certain

activity

cells

incubated

neither

tubulin

inhibit

catalyzed

specific

treatments

a concentration

also

one-third.

in

the the

low

ligase

activator

brain

they is

by

in the reported

is

which

reduced

an

rat

tubulin Data are

incorporation

medium

Furthermore, by

incorporated

tubulin:tyrosine

or

A by measuring tubulin:tyrosine

purified

13H]-tyrosine

medium

is

of

3x cycled

of

of

of

the

tyrosine

Extracts

brain

ligase

in

CHO cells

tyrosination

amount

pattern

0.03

carboxypeptidase highly

h conditioned of

an

with

difference between total tyrosinable

Twenty-four

epithelial-like

synthesis

the the

pulsed

concentration

with

the

This

bu2cAMP-testosterone.

the

treatment incubation

measuring A.

calculated binding determinations.

evidence

the

including

after during

morphology.

the

endogenous

tyrosine

3.9

h (Fig.

purified

It

1.4

CHO

fed”

1 h with

tein

f

tubulin by colchicine 4 separate

24

is

for

the

24.3

is determined by with carboxypeptidase

e.g.,

There

highly

6.3

epithelial-like

nutrients,

in

pmolshg soluble protein

f

a fibroblast-like in

% of total tubul in

110.3

90.7

Total tyrosinable the amount of 1 igase.

assumed

pmlshg soluble protein

with

min

Pulsed conditioned

under

Non-Tyrosinated

pmols/mg soluble protein

for

Fractions

Conditions

Tyrosinated

Pre-incubated buzcAMP-testosterone

COMMUNICATIONS

activity we

that

by

of

employed,

inhibits

pro-

99%.

likely,

then,

results

from

that the

the rapid

1647

activation

of

carboxyl

generation

of

tyrosinable

a

terminal substrate

BIOCHEMICAL

Vol. 100, No. 4,198l

stimulated

by

conditions

mation).

It

protein

synthesis.

is

synthesized boxy1

in

1 h is

the

can

carboxyl generated

prepared

by from

terone

for

at

carboxy

the

nor

could

be

CAMP-stimulated cells

into

Table

1.

formation

does

experiments,

conversion

of

carboxyl

de of

at

the

de

nova

proat

substrate Extracts

with

of

car-

tyrosinated

activity. incubated

no~o tubulin

tytosinable

calf

bu2cAHP-testos-

brain

cell

types

is

tubulin

labeled

soluble

of

is

to

as

possibility by

that

there

some

yet

these

shown

reverse

much

a

in

in

trans-

tyrosinable

varies

tubulin

by

tubulin

lead

amount

tyrosinable

perhaps

fractions

tubulin the

tyrosinable

pool,

that

the

tyrosinated

that

of

conditions

exclude

neither

therefore,

non-tyrosinable

tyrosinable

to

3).

It

under has

tyrosine cells

the

conditions

abruptly

stops

that

otherwise

impact

on

CHO cells

metabolism

(Fig.

3).

Thus,

resembles in

and

a similar

myogenic

Podophyllotoxin inhibits

amount

tubulin that

distribution and

cannot

inhibits

terminus

a tubulin

by

tubulin. as

+ 6% is

in

a slow

unknown

post-

modification.

Podophyllotoxin

CHO

the

a non-tyrosinable

increase

non-tyrosinable

translational

of appear

many

such

under

of

we

it

possible,

The

incubation

amount

separate

on

seemed

tyrosinable

the

the

transfor-

tyrosinated

detyrosination

in

from

significantly

since

terminal

It

not

70%

CHO cells

tubulin

generated

term

However,

(Fig.

of

phosphorylation.

Short

cells,

(reverse

[ 3 H]-tyrosine.

with

tyrosinated,

accomplished

COMMUNICATIONS

bu2cAMP-testosterone,

does

and the

(3,4,20).

is

a carboxypeptidase-like

cells

catalyze

shape

a tubulin

about

Nor of

amount

tyrosinable

substrate

activation

not

the with

only

1).

control

of

RESEARCH

cell

this

treated

treated

for

in

that

2.5%

cells

terminus

A,significant

than

(Fig.

both

changes

cycloheximide

account

an

1 h do

the

in

terminus

to

however,

less

Even

synthesis

lead

possible, In

terminus.

tein

is

not

that

AND BIOPHYSICAL

its cultures interrupts

generation

of

impact

on

tyrosine induce that the

carboxyl

incorporation reverse

have effect

terminal

a of

at transformation

limited

carboxyl

podophyllotoxin

tyrosine

metabolism

(5). microtubule tyrosinable

treadmill substrate,

1648

metabolism an

effect

the

that

(23)

and

is

apparent

of

BIOCHEMICAL

Vol. 100, No. 4,198l

long

before

microtubules

detyrosination

is

release

of

of

from

(8).

treadmi

11

and

the

from

resulting

in

dimer

pool.

result

in

only

an

increase

microtubules

fibroblast-1

conditioned

medium. that

milling

lead

The to

that

to

stimulation sponse response

nerve

tyrosine to

is

apparent

metabolism, a variety

the

of

environmental

activity

the

anchor

those

neurites

the

cells initial

1 min may

result

from

pulsed

have

(25). where

The the

an

when

growth

tri

factor, but

only

of

the

the

h

early of

tread

immediate

and

turated,

more

modest

morphological cell

activation

of

initial

response

re-

membrane, carboxyl of

a termi-

the

stimuli. ACKNOWLEDGEMENTS

We are grateful to Dr. R.C. Henneberry for the gift of the CHO-Kl cells, to R.K. Hanson and A.M. Obuchowski for technical assistance, and to J.C. Hobbs and A.M. Obuchowski for help in the preparation of the manuscript. This work was supported in part by Grant NS14240 from the National Institutes of Health.

1649

24

a procedure

A rapid, the

the

cytoskeleton.

noted

metabolism nerve

in

number

the

the

reverse

during

of

If

explain

fully

a greater

ruffling

(26).

the

occurs

involve

we

could

cells

that

of

unprimed is

in

not

tubulin.

this

observed in

would

tyrosinated

points,

tyrosine

of

within then,

treadmilling

reorganization

presence

in

factor

tyrosinated

may

terminal

observed

the

PC 12 cells,

regeneration

growth

that

with

in

and

as

the

tyro-

microtubules

incorporation

during

metabolism

[ 3HI-labeled end

not release

microtubule

of

transformation

replated

also

well

the

the

tyrosine

as

continuing

carboxyl

a rapid is

to

1,

experiments

are

leads

ccl

reverse

of

cells

from

in

treadmill

plus

the are

of

activity

of

released

stimulation

primed

specific released

ike

preliminary

major

brane

are

microtubules In

the

continued

incorporation

transformed

nal

in

self-limiting

events

1 ibration,

these

that

do

result

substrate. the

and

detyrosination

initiating

at

that

microtubules may

tyrosinable

between

equi

and

anchored

thereby

incorporated

equilibration

After

a few

rapid,

an

microtubule that

points of

be

the

proposed

metabolism

bu2cAMP-testosterone

anchor

then

of

COMMUNICATIONS

We have

treadmill

suggested

with

such

could

end

has

generation

dimers

microtubule

minus

Treatment

subsequent

sinated

the

RESEARCH

(8).

disassembled

upon

Kirschner

(24).

microtubules

fully

dependent

dimers

coordinated

are

AND BIOPHYSICAL

mem-

Vol. 100, No. 4,19Bl

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES

1.

Deanin,

G.G.,

Thompson,

W.C.,

and

Gordon,

M.W.

(1977)

Dev.

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Pierce, T., Hanson, R.K., Deanin, G.G., (1978) in Maturation of Neurotransmission, pp. 142-151, Karger, Base?. Rodriguez, J.A., and Borisy, G. (1978)

Gordon, M.W., Vernadakis Biochem.

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Barra, H., Arce, 439-446. Thompson, W.C., Sci. U.S.A. 76, Bothwell, M-A.,

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22. 23. 24. 25. 26.

Nath, J., and Flavin, M. (1979) J. Biol. Chem. 254, Hynes, R.O., Destree, A.T., Mautner, V.M., and Al i, Supramol. Struct. 7, 397-408. Preston, S.F., Deanin, G.G., Hanson, R.K., and Gordon, Mol. Evol. 13, 233-244. Margolis, R.L. (1981) Proc. Natl. Acad. Sci. U.S.A. Kirschner, M.W. (1980) J. Cell Biol. 86, 330-334. and Black, M.M. (1980) Greene, L.A., Burstein, D.E., in Neurobiology, Giacobini et al. eds., pp. 313-319, York. Connolly, J.L., Greene, L.A., Viscarello, R.R., and J. Cell Biol. 82, 820-827.

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in Tissue Culture Raven Press, New Riley,

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