Vol. 81, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 30, 1978

Pages 344-350

RNA TUMOR VIRUS PHOSPHOPROTEINS: PHOSPHORYLATION OF PRECURSOR AND PROCESSED POLYPEPTIDES Bijay K. Pal and Pradip Roy-Burman Departments of Pathology and Biochemistry University of Southern California School of Medicine Los Angeles, California 90033

Received

January

2O,L978

Summary: Monospecific antisera made against the 30,000 molecular weight major internal polypeptide (~30) and the 12,000 molecular weight phosphorylated polypeptide (~~12) of a wild mouse type C oncovirus were used to imnunoprecipitate precursor polypeptides from extracts of isotopically labeled cells infected with the oncovirus. Analysis of the immunoprecipitates by SDSpolyacrylamide el electrophoresis led to the detection of several precursor polypeptide (Pr B species containing the determinants of both p30 and ppl2. These species, namely Pr>lOO, PrlOO, Pr77, Pr62, and Pr50, were all found to be phosphorylated in pulse experiments. The polypeptide ppl2 was, however, the major phosphorylated species immunoprecipitated by anti-pp12 sera in pulse and chase experiments. These data and a relatively high degree of phosphorylation in the processed ppl2 suggested that phosphorylation of oncovirus protein is initiated at the polyprotein level and the phosphoprotein moiety is further phosphorylated subsequent to processing. Mature

viral

proteins

cleavage

of intracellular

leukemia

virus,

tified

(4-6).

tides

containing

ranges for

termed

low molecular ~30,

~15,

species

~12 is

primate

viruses

~12,

phosphorylated is ppl5

one of the gag proteins interest

that

protein

processing

of the

we present

results

murine undergoes

leukemia further

(Pr55

(10,000

to 30,000

and ~10.

In type (ppl2),

(8-10)

whereas

that virus

are

the

viral

and that

phosphorylation.

0006-291x/78/0812-0344$01,00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

it

is ppl9 it

344

proteins

mammalian

phosphoprotein

is

gag proteins.

gag precursors

phosphorylated

intracellular

lower

may have a significant

to the mature

show that

The gag gene codes

the analogous viruses

(gag)

structural

by phosphorylation,

phosphorylation

precursors

(7).

of the

iden-

of the polypepantigens

daltons)

murine

have been

size

to Pr200)

C viruses

and in avian

is modified

polypeptides

group-specific

daltons

by the

In Rauscher

the approximate

of the viral

weight

are formed

(l-3).

precursor

precursors,

to 200,000

viruses

polyproteins

virus-specific

determinants

55,000

C RNA tumor

precursor

Among these

from

four

several

of type

of a wild the processed

(11).

in Since

of considerable role

in the In this

paper

mouse strain pp12 product

of

Vol.81,No.2,1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATiONS

MATERIALS AND METHODS Cells and viruses. Feral mouse embryo cell line SC-l (13) chronically infected with wild mouse (WM) 292 virus (14) was maintained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Cells were grown in 60 mm2 Petri dishes to near confluency, washed three times with warm Earle's balanced salt solution (EBSS) and labeled with C3H]-amino acid mixture (10 uCi/ml) and [32P]-phosphate (250 &i/ml) in EBSS. Cells were incubated for 10 min in pulse experiments and 10 min followed by 2 hr chase in complete growth medium (MEM containing 10% FBS) in pulse-chase experiments. After incubation the medium was removed, the cells were washed twice with ice cold EBSS, and then immediately collected by scraping. An NP-40-deoxycholate lysate of the cells was prepared by a modification of the procedure of Vogt and Eisenman (1) with the exception that protease inhibitor, phenylmethylsulfonylfluoride, was added at a concentration of 300 pg/ml (15). The cell lysate was then centrifuged at 15,000 g for 15 min to obtain the supernatant (cell extract) for the immunoprecipitation experiments. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Specific antisera against gel filtration purified proteins (16), p30 and pp12, of WM292 virus were made in New Zealand white rabbits as described (17). Immunoprecipitation reactions were carried out by mixing cell extracts from pulse or pulse-chase experiments, with the optimal amount of specific anti-p30 or antipp12 serum. The mixture was incubated at 37" for 15 min and then 16 to 18 hr at 4". Immunoprecipitates formed were collected by centrifugation at 2000 g for 20 min at 4O, washed three times with ice-cold STE (100 mM NaCl, 10 mM Tris-HCl and 1 mM EDTA, pH 7.4), and analyzed by SDS-polyacrylamide gel electrophoresis (16, 18). Standard proteins, bovine serum albumin, ovalbumin, chymotrypsinogen A and cytochrome C were included in identical gels as molecular weight markers. Gels containing marker proteins were fixed and stained with Coomassie blue and those containing radioactive samples were fractionated by slicing and the radioactivity in the slices determined by liquid scintillation counting (19). RESULTS Specificity

of WM-292 virus

the

specificity

40,

and radioiodinated

with

of the antisera,

the anti-p30

These

and anti-pp12

did

Precursor cells,

gels

and possess antisera

pulse

phosphate,

were

tates

were

studied

(Fig.

2A) show that

purified

WM-292 virus

sera.

show that

mutually not react

for

method Analyses

the anti-p30

exclusive with

phosphorylation. labeled

and anti-pp12

by the chloramine-T

SDS-polyacrylamide specific

anti-p30

Extracts

10 minutes

imnunoprecipitated

with with

by SDS-polyacrylamide several

of the

SC-l

acid sera

(Fig.

both

1).

cells.

mixture and the

in

are mono-

determinants

electrophoresis.

containing

345

sera

from WM-292 virus-infected

anti-pp12

NP-

immunoprecipitates

and anti-pp12

r3H]-amino

with

and immunoprecipitated

of uninfected

gel

polypeptides

To determine

was disrupted

(20),

immunological

extracts

sera.

SC-l and [32P]immunoprecipi-

The results [3H]

and [s2P]-labels

Vol. 81, No. 2, 1978

BKXHEAKAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8

% -

I

x t 0

60

0 1

IO

20 FRACTION

30 40 NUMBER

30

02

IO

FRA%ON

i%MBER

40

so

Fig.

1.

Specificity of anti-p30 and anti-pp12 sera. Immunoprecipitates obtained from radioiodinated disrupted WM-292 virus with anti-p30 sera (A), and with anti-pp12 sera (B), were electrophoresed in 7.5% SDS-polyacrylamide gels.

Fig.

2.

SDS-polyacrylamide gel electrophoresis of imnunoprecipitates obtained with antisera against WM-292 virus pp12. A. Immunoprecipitate formed with extract of virus infected cells pulse-labeled for 10 min. with C3H]-amino acid mixture and [s2P]-phosphate. B. Immunoprecipitate obtained from extract of cells pulse-labeled for 10 min and then chased for 2 hr in unlabeled growth medium.

were

resolved.

they were

Since

considered

imate

molecular

Pr62,

Pr50,

labeled

anti-pp12

weights,

and Pr35.

were higher

polypeptide

species

to be precursors the

pulse.

phosphorylated amounts

serum precipitated

of this

species

The mature

in such a short

polypeptides relatively

these

were

viral It

contained protein.

designated polypeptide,

should and that

of phosphate

(Fig.

the mature

346

Based on their as Pr>lOO, pp12,

be noted Pr>lOO

pp12 determinants,

that

all

and Pr62

2A).

pp12 protein

PrlOO,

however,

approxPr77,

was not

the precursor species

In pulse-chase in addition

contained experiments, to the

pre-

Vol. 81,No.2,

Fig.

3.

cursors

described

above

(Fig.

pp12 whereas one.

4

4

2

2

(Fig.

that

the

for

the phosphate

higher.

These subsequent Figure

The ratio a major

that

content

data

Although

major

that

to cleavage

from

precursor,

such as Pr62,

results

processed

only size

with

precursor anti-p30

347

at the

pp12

was less

between at least

is further

was

20 for

pp12 moiety,

difference

pp12 was still

the processed the

of the pp12 species was about

the molecular

of the

species

to [3H)-counts

phosphorylated

for

precursor

of phosphorylation of [32P]

indicated

3 shows our

all

Pr62 was phosphorylated

C3H]-counts

pp12,

2B).

the extent

2B).

Assuming

normalizing

lated

6

phosphorylated,

highest

than

6

SDS-polyacrylamide gel electrophoresis of immunoprecipitates obtained with antisera against WM-292 virus ~30. A. Imnunoprecipitate formed with extract of virus infected cells pulse-labeled for 10 min with C3H]-amino acid mixture and [32P]-phosphate. 8. Immunoprecipitate formed with extract of cells pulse labeled for 10 min followed by 2 hr chase in unlabeled growth medium.

remained the

1978

and

Pr62 and four-fold

phosphory-

polypeptide. sera.

This

sera

recognized

all

Vol. 81, No. 2, 1978

the precursor

species

amount

of PrlOO

visible

in the

3B). well

resolved

of the tion

pulse

(Fig. mature

was highest

resolved

experiments

In addition,

of the various

anti-p30

it

(Fig.

Pr62,

(Fig.

indicates

the

noted

was 3A and

and Pr50,

were

nonphos-

38).

that

with

was also

The small

precipitated

experiment

and lowest

of phosphorylation

sera

Pr77,

also

precursors

in the case of Pr>lOO

Pr35. although

anti-p30

Pr>lOO,

in the chase

except

experiments,

using

precursors,

p30 formed

sera

in the pulse

and chase

3A).

in the degree

in Figure

by the anti-pp12

phosphorylated

32P/sH ratio

ation

detected

was not

Four major

phorylated

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A comparison

the phosphorylaPr77.

Such vari-

in experiments

described

2. DISCUSSION

The results polypeptides,

in this Pr>lOO,

report

Pr77,

show that

Pr62,

and Pr50,

the gag gene proteins,

p30 and pp12,

very

precursors

likely

Pr80,

that

Pr65,

leukemia

and Pr55

(7).

Another

of pp12 but not

~30,

cells

of different

peptide

whose order

Since

Pr45 does not part,

intracellular

while

portions

contain Pr35

However,

Pr45

(7),

gag-gene

Whether

Pr35,

this

at various virion

which

(7),

it

probably

contains

observation

is

sites

remains

or intracellular

348

poly(21).

that

the

all

The degree being

related

of the precursors

repre-

represents

We find

and Pr62

the

mouse

precursor

are phosphorylated. Pr>lOO

murine

to their

coded

polyprotein,

of

relatively

to sequential

at the pp12 moiety

unclarified

is

containing

in the wild

may be the pl5-pl2-fraction.

seems to vary,

the stable

(Pr35),

may be related

p15 determinants

It Pr200,

as NH,-p15-p12-p30-plO-COOH

and dephosphorylation

due to phosphorylation

from

of the murine

including

virus.

in Rauscher

polypeptide

difference

with

to the corresponding

we have detected

has been determined

determinants

detected

different

This

however,

phosphorylated.

phosphorylation

study.

apparently

precursors,

phosphorylation, highly

is

that

precursor

mouse leukemia

equivalent

cells

infected

p30 and pp12 moieties.

p12-p30-

are

intracellular antigenic

of the wild

polyproteins

virus-infected

sentation

share

gag precursor

virus

determinants

both

these

the major

or

in the present P60,

has been

Vol.81,

No.2,

reported levels

to be phosphorylated of phosphorylation

been detected detectably

after

examine phorylated

that

cleavage

whether or

all if

as in the mature possible

role

they

Pr65

from the

results

viral

the precursor

also

exist

extracellular

cited

is further

mature

virion

(9,

of the gag precursor

polypeptides

It would

22, 23).

of phosphorylation/dephosphorylation are

being

our in the

are equally

phosphorylated This

7).

be important

aspect

in the cleavage currently

number

phosphorylated

multiple

10,

is not

phosphorylation,

pp12 molecules

in non-random

Pr80

in reference

polypeptide.

intracellular

Differential have also

whereas

of precursor

pp12

(12).

polypeptides

is phosphorylated

in the level

the mature

RESEARCH COMMUNICATiONS

on the pp12 moiety

(unpublished

to the variation suggest

primarily

authors;

phosphorylated

results

AND BlOPHYSlCAL

of the mouse gag precursor

by other

In addition

cell

BIOCHEMICAL

1978

to phos-

species and the processes

investigated.

ACKNOWLEDGEMENTS the

This Virus

investigation was supported by contract number NO1 CP 53-500 Cancer Program of the National Cancer Institute.

within

REFERENCES 1. 2. i: 5. 6. 7. 8. 9. 10. i:: ::: E:

Vogt, V. M. and R. Eisenman. (1973) Proc. Natl. Acad. Sci. USA 70, 1734-1738. Naso, R. B., L. J. Arcement and R. B. Arlinghaus. (1975) Cell 4, 3136. Okasinski, G. F. and L. J. Velicer. (1976) J. Virol. 20, 96-106. Arcement, L. J., W. L. Karshin, R. B. Naso, G. A. Jamjoom and R. B. Arlinghaus. (1976) Virology 69, 763-774. Van Zaane, D., A. L. J. Gielkens, M. J. A. Dekker-Michielsen and H. P. J. Bloemers. (1975) Virology 67, 544-552. Jamjoom, G. A., R. B. Naso and R. B. Arlinghaus. (1977) Virology 78, 11-34, Arcement, L. J., W. L. Karshin, R. B. Naso and R. B. Arlinghaus. (1977) Virology 81, 284-297. Pal, B. K. and P. Roy-Burman. (1975) J. Virol. l5-, 540-549. Pal, B. K., R. M. McAllister, M. B. Gardner and P. Roy-Burman. (1975) J. Virol. 16, 123-131. Hayman, E. G., B. K. Pal and P. Roy-Burman. (1977) J. Gen. Virol. 3&, 459-469. Lai, M. M. C. (1976) Virology 74, 287-301. Oskarsson, M. K., C. W. Long, W. G. Robey, M. A. Scherer and G. F. Vande Woude. (1977) J. Virol. 23, 196-204. Hartley, J. W. and W. P. Rowe. (1975) Virology 65-, 128-134. Gardner, M. B., B. E. Henderson, J. D. Estes, R. W. Rongey, J. Casagrande, M. Pike and R. J. Huebner. (1976) Cancer Res. 36, 574-581. Fahrney, D. E. and A. M. Gold. (1963) J. Amer. Chem. Sot. 85, 997-1000. Pal, 8. K., M. Wright, J. E. Officer, M. B. Gardner and P. Roy-Burman. (1973) Virology 56, 189-197.

349

Votz 81, No. 2, 1978

17. 18. 19. 20. 21. 22. 23.

BIOCHEMICAL

AND B0PHYSlCAt

RESEARCH COMMUNICATIONS

Strand, M. and J. T. August. (1973) J. Biol. Chem. 248, 5627-5633. Summers, D. F., J. V. Maize1 and J. E. Darnell. (196r Proc. Natl. Acad. Sci. USA !&, 505-513. Roy-But-man, P., B. K. Pal, M. B. Gardner and R. M. McAllister. (1974) Biochem. Biophys. Res. Comnun. 56, 543-551. Greenwood, F. C., W. M. Hunter and J. S. Glover. (1963) Bi ochem. J. E, 114-123. Barbacid, M., J. R. Stephenson and S. A. Aaronson. (1976) Nature 262, 554-559. Hayman, E. G., B. K. Pal, M. M. C. Lai and P. Roy-Burman. ( 1977) Biochem. Biophys. Res. Connnun. 78, 1156-1161. Pal, B. K. and P. Roy-Burman. n977) Virology 83, 423-427

350