Novel characteristics of a 33KDa protein (pp33) rapidly phosphorylated in IL3 dependent cells by stimulation with IL3

Vol. 164, No. 1, 1989 October

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

16, 1989

lBOVEL

Pages

OF

CHARACTERISTICS

PBOSPHORYLATED

John

lDept.

MGarland

IB

IL3

I,*

, Douglas

Cell

(L Structural

2Programme Development

Resources Program,

3 Laboratory Response

Received

of Modifiers

September

A

33KDa

DBPEEDRET

PROTEIN

CELLS

K.

BY

Ferris2

Biology,

(pp33>

RAPIDLY

STII’IULATIOB

and

VITH

William

University

of

Biological Inc. , ESP., Frederick Cancer Frederick MD 21701

520-530

IL3

L. Farrar3 Manchester,

U.K.

Carcinogenesis research Facility,

Molecular Immunoregulation, Program, Frederick Facility, Frederick MD 21'701

Cancer

&

Biological research

6, 1989

We report the novel properties of a 33 KDa cellular protein rapidly phosphorylated by stimulation of growth by IL3 in IL3 dependent lines. Although pp33 is readily soluble in SDS, SDSnon-ionic detergents and is solubilised pp33 is insoluble in excluded from electrophoretic analysis (IEF, NEPHGE) employing such detergents. Native pp33 is not extracted by non-ionic detergents with or without cation chelaton. pp33 is concentrated in a cell containing endoplasmic fraction reticulum where it is associated with a specific ti-ypsinsensitive degredative enzyme, active at 4 degrees. Its unusual characteristics and kinetics of phosphorylation suggest pp33 its absence in may be a novel molecule, explain studies elsewhere where non-ionic detergent extraction has been exclusively used and suggest it is intimately related to the signal transduced by IL3. 4' 1989 AcA,ivn, i'l
Activation

of

protein

transduction(l-4). haematopoeitic cc-fms)

been

for

"Address

of

KDa for

klnase

a number

in

known

about the

3

(IL31

of

other proteins

moeities with

MW's

CSF-1 (5),

has

stimulation

or

signal

activity

Interleukin IL3

event

Although

Nevertheless, with

key is

tyrosine

multipoetin

cells

phosphorylation 60-160

little signaling.

characterised.

dependent

a

factor

possesses

the

is

However, growth

receptor receptor

kinases

the

not of

yet IL3-

results ranging

in from

(6-13). correspondence:

Department

of

University,

Oxford

Cell Road,

0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

J. &

M.

Garland,

Structural

Manchester

520

Division Biology,

Ml3

QPT,UK.

of

Anatomy,

Manchester

Vol. 164, No. 1, 1989

BIOCHEMICAL

Recently, 33

KDa

very

protein,

Phosphorylation

of or

factors

(but

in to

SDS

correlation on

cell

forming

of

metabolism an

for

integral

pp33

methods

by 16)

part

suggest of

been have

the

The

non-ionic

detergents or

Further, non-ionic

phosphorylation

(15,

not

isofocussing

chelation.

precipitated

is IL3-

behaviour. in

samples

and

has

extraction

calcium

is

pp33

c

growth

growth)

since

insoluble

prepare

other

spontaneous,

electrophoretic

is

without

in

close

protein

to

or

solubilised

its

pp33

used

However,

two

kinase

by

active a

signal

SDS-

PP33

detergents. with

it

a

(14).

protein

mediated

various

analyse

that

with

IL3

be in

studies,

in lines

to

stimulating

clone.

other

show

commonly

of

those

malignant

used

The

insensitive can

of

demonstrated dependent

phosphorylated

described

PAGE,

is

phosphorylation

been IL3

pp33

only

independent been

has

inhibition,

constitutively

results

IL3-dependent

PP33, different

physiologically stimulation

rapid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

may

be

transduced

effects a novel by

IL3. Haterials

and

Hethods

Cell lines. The murine dependent lines AC2 (sub clone RB5), 123, FDC-Pl and NFS 60.8 were maintained as previously described <14). Recombinant IL3 was used in experiments. with =-P. <)Labelling with phosphorus was as Labelling 1 (-Jc-. (2 x previously described (14). Briefly, 107 were washed free of IL3, incubated exponential phase cells min for specific times in RPMI/5%FCS, followed by 30 incubation in phosphate-free RPMI/X% FCS (dialysed against O.OlH CaCl, O.lW glucose, O.OlM KCl, 1.4M choline chloride, Cells were resuspended to 5 x 10" to 0.2M HEPES). O.OlM MgCl, 5 x 10" per ml in phosphateand serum-free RPMI, 0.5 ml Eppendorf tubes and the cells pre-labelled for distributed to IL3 was 15 min with 200-400 uCi/ml phosphate at 37 degrees. and after known intervals the cells then added (10-100 U/ml>, removed and pellets down set) , the supernates spun (10 addition of 250 UL 10% TCA; or precipitated by direct detergent extraction buffer and extracted in 100-300 UL room temperature or 4 degrees with extracted 10 - 60 min at controlControl cells received equal volumes of mixing. vectored cos supernates or medium. TCA precipitates were washed once in PBS and Electrophoresis. spun in distilled water. Aliquots were and pellets once extraction Laemmli sample buffer. Detergent dissolved in 20mM Tris HCl pH 7.41 for 2D electrophoresis, buffers were: 50mM NaCl/ 0.5% NP40/ 0.5% sodium deoxycholate/ 4mM iodoacetic for acid/ 5mM sodium pyrophosphate/ 5mM NaF and 6M urea; 0.1 - 10% NP40, Triton xl00 single-dimension electrophoresis, or octyl glucoside/ 1OmM Tris HCl pH 7.11 5mM NaF and 5mM 1OmM EDTA. Detergent sodium orthovanadate, with or without extracts were spun 8OOOg x 30 min and both supernates and

521

BIOCHEMICAL

Vol. 164, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In some experiments, pellets processed for IEF or SDS-PAGE. washed solubilised in the non-ionic TCA precipitates were Samples for SDS-PAGE were reduced with ZME detergent buffers. reducing SDS-PAGE with boiling before electrophoresis in Laemmli gels. IEF or NEPHAGE first-dimension gels were run in followed the presence of 6M urea in 1.5mm x 12 cm tube gels, by reducing SDS-PAGE in the second dimension, cells were Intracellular distribution of Labelled PP=immediately diluted by addition of an equal volume of icecold Tris HCl pH 7.4 containing 1mM NaF/lmM sodium orthovanadate and lOug/ml PMSF. Suspensions were sonicated on ice with 6 x 1 set pulses of ultrasound, checked for breakage and spun at various speeds at 4 degrees. Pellets and samples supernates from each spin were precipitated with TCA or of solubilised directly In Laemmli sample buffer for SDS-PAGE For trypsin treatment, analysis. the 100,000 g x 1 hr pellets were suspended in 100-300 UL 1OmM Tris HCl buffer pH 7.6 or PBS containing various concentrations of trypsin and incubated on ice for the times shown. Trypsin inhibitor was added in lo-fold excess and the suspensions respun at 100,OOOg x 1 hr. Pellets were solubilised in Laemmli buffer, supernates either TCA precipitated or mixed with 10x concentrated Laemmli buffer for direct electrophoresis. Phospho-amino-acid identified analysis. Bands from autoradiographs were excised from the extracted with gels, 100-300 uL 50 mM ammonium carbonate/ 0.1% SDS for 15 min2-mecapto-ethanol added to 5% and the gel fragment boiled 10 min, followed by extraction at 37 for 24 hr. Gel iragments were removed, the tubes dried under vacuum and residues washed 3 times with 100-300 UL ether-alcohol 1~3 and dried. Samples were hydrolysed with 6N HCl for 1 to 1% hr at 110 degrees, dried, resolubilsed in water and electrophoresed on cellulose acetate plates in pyridine:acetic-acid:water 1: lo: loo. Phospho-amino-acids were identified by incorporation of known standards in the samples identified by ninhydrin and autoradiography. Chemicals and growth factors. Recombinant IL3 (2 x 10".,U/ml in serum-free cos supernates) was a kind gift of Dr. T. Yokota, DNAX Laboratories, Palo Alto, California. Purified IL3 (2 x 105“ U/ml> was a kind gift of Drs. Keller and Ruscetti, NCI, Alcoholand carrier-free isotopes were obtained Frederick, from Amersham International. Ampholines were obtained from LKB-Pharmacia.

Results Early

changes

in

IL3

stimulated

TCA

precipitates,

shown).

To

pp33, quickly

cells

mln minj;

as and

then controls

pp33 the

on

IL3

same (fig.11

determine were possible

the

stimulated were

in

in with

starved

of

SDS

IL3 of

522

15 (total IL3

recombinant in

extracts

phosphate

phosphate-free over

and

identified

direct

rapidity

washes

Pure

phosphorylation

and

washed (3

starvation.

pp33


exchange

on

conditions

min),

prelabelled

starvation throughout.

as for

time Samples

of

10 25 were

Vol. 164, No. 1, 1969

BIOCHEMICAL

rB5

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

123

I

-27

-17

C

rm.

P

I

C rm.

P

p33 Recombinant and Purified IL-3 Stimulation of pp33 by purified and recombinant Fig. 1. Cells were starved for 1 hr, and stimulated for Controls received an amount of PBS equal to the volume Prestained MW standards. rm = recombinant IL3; IL3. IL3. Arrow marks pp33. TCA precipitation/SDS-PAGE.

removed in

at

1,

cells

5

control pp33

virtually

1>,

from

changes stable

hydrolysis

of was

the

on

IL3

ratio

cells

min

not

depleted immediately demonstrated

labelled

by

shown). IL3

that

phosphotyrosine 523

over

to

the

the

for is

10

initial

presence

Since to

on Acid

incorporated was

is

periods

occur

withdrawn. all

of

pp33

longer

appear IL3

that

phosphorylation

equal

of

protein

2 shows

five-fold

remained

in pp33

pp33 and

unaffected

phosphorylation

Xo

Fig.

the

5

(data

excised serine.

SDS-PAGE.

was

(50ug/ml)

relatively label

with within

cells

absent fig

for

phosphorylatlon

cycloheximide
min.

increased

in

value.

10

supplemented

significantly min;

and

IL3. min. pure pure

15 of P =

detected.

a

BIOCHEMICAL

Vol. 164, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4

yj

0.469 1 140.96

80.00

IO.00

140.96

+IL 3, 1 min

-IL

2.222

2.156

0.567

0.567

3, 1 min

+ IL 3, 5 min

0.725

LO.00

140.96

+IL 3,10

80.00

140.96

min

3, 10 min

-IL

of p33 phosphorylation in RB5 cells Fip;. 2. Microdensitomtry stimulated by IL3 immediately after washing. Total starvation time including washes and prelabelling was 25 min. Arrow = A = reference peak. Ordinate = optical density. Note pp33; overall increase in phosphorylation in all samples, and relative increase in pp33 in stimulated cells. Similar results were recorded for 123 cells. TCA precipitation/SDS-PAGE.

2-diensional IEF

electrophoresis.

of

cells

extracted

precipitates and

were

2D

IEF

PAGE,

it

NEPHAGE that

presence

Solubility demonstrated

not

detected

NEPHAGE

of of by

insoluble

phases

detergent


in

pp33

the

was

IEF in

identified

was

pH

3-10

sample

in

4-7

IEF

second-dimension from

TCA

SDS-PAGE

conspicuous or

i'D

buffer. by

gels NEPHAGE

SDSor

gels

first-dimension

excluded

in

parallel

pp33 in

However,

was

in

analysed Although

remained that

pp33

directly

therefore

NEPHAGE.

gels.

indicating the

and was

pp33

No

in

showed (fig.

gels

3) (

even

in

tritoniurea.

PP33 TCA-SDS of

the (fig.

non-ionic

in was

detergents.

absent 4).

from cells

same

Similar 524

both

pp33

the

soluble

extracted

in

results

were

non-ionic obtained

and

Vol. 164, No. 1, 1989

neplxage

a

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b

+IL3

P

IEPHAGE electrophoresis of TCA precipitates from RR5 Fig. 3. stimulated 15 min. (3a) or cells starved 1 hr. and Second dimension SDS/PAGE. A small amount unstfmulated (3b). of pp33 appears as a streak
with

NP40

and

concentrations

(O.l%,

partial

by

1%

5;

by

addition

of

pp33

Distribution enzyme.

Nearly

pellet, high-speed

and

absent

from Further-

low

an

of

with

a

degredative

enzyme

a

in

that

trypsin.

Thus

highly

of

or

g

x

1

hr of

pp33

specifically

pp33

pp33

active,

(phosphatase

degredative

whereas

was buffer

loss

is fact

4 degrees)

but it

A

cytoskeletal

and

pp33,

incubated

in

a

and

trypsin,

pp33

is

100,000

ug/ml

release

of

detergent.

with in

(50

showed

concentrations

associated

non-ionic

reticulum

to

EDTA.

SDS

association

tubes

experiments

of

appeared

containing

control

without

in

digestion

different

absence

excess

its

pp33

failed

tubes

or

solubilised

of

trypsin

pellets in

with

three

complete

endoplasmic

Mild

persisted

at

10%)

the

pp33

all

containing

fragments.

and

for

fig.

precipitated

by

glucoside

explanation

provided

BT.

octyl

in

on

ice

is

protected

situ

(fig. appears

trypsln-sensitive

hydrolase)

active

at

low

temperatures. Discussion insoluble used be

Our from

to

prepare

enzymically

extraction

unusual

SDS

finding

solution

samples

by for

degraded buffers;

thus

that

non-ionic

pp33

low absence

525

is

rendered

detergents

commonly

Further,

electrophoresis. at

the

is

temperatures of

pp33

it in

in

studies

may these using

Vol.

164,

No.

1, 1989

BIOCHEMICAL

AND

1 hr ’

RESEARCH

COMMUNICATIONS

1 hr

rB5 +

BIOPHYSICAL

,

123’ - I +

rB5 +

-

1 hr 123

- I +

.

xi---+-+-

-

123 l

75-

5050-

39-

27-

27-

27-

TCA ppts Fig. Cells Arrow

4. were

Detergent

Distribution starved

of for

1

Pellets

pp33 in hr and

Detergent

TCA and stimulated

detergent 15 min

SN

extracts. with IL3.

= pp33. + = with IL3, - = without IL3. pp33 is present in the TCA precipitates of both RI35 and 123 cells stimulated Some pp33 is present in the detergent pellet from with IL3. 123 cells but absent from RI35 cells. Ho pp33 appears in any supernates.

non-ionic

detergent

phosphate

exchange

can

be

detected

Possible

on

proteins not

histones

cdc-2

rapidly

specific released

are

(20,21)

is

may

by

be

kinases

and the

IL3),

526

KDa

S6 is

cycle

that

Although

and serine

KDaJ

regulated

is both

by would

S6

(27).

cdc-

(19)

(30-32

(22-26))

phase actin-binding

cell

show calpains

excluded. and

detergent-soluble calcium and

detergent-soluble

32

protein phosphatase

changes

since

studies by

factors a

as

the

(preliminary

ribosomal

Finally,

removed.

reasonably

growth

rapid

such

PP33 (17),

(18)

of

into membrane-bound

IL3

36KDa)

explained.

be

extremely

phosphorylated

phosphorylation stimulated

may is

for (MW

cyclin protein

is

pp33

immediately

candidates

control 2

extractions

be

Lipocortins

proteins

of

M-W

Vol. 164, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

27-

J

+-+-

-

rB5

+

rB5

123

Dlalysed Against Trim After TCAISDS

TCAISDS

Fig. 5. Precipitation of pp33 by triton Xl00 after solubllisation in SDS (see text). Arrow = pp33. pp33 appears in the original TCA/SDS solution (left) and in the precipitate formed after addition of excess triton to SDS-solubilised pp33 (right). No pp33 was detected in Triton-soluble supernates (not shown). + = with IL3, - = without IL3.

36-40

which

KDa,

are

substrates

and

sarc

ionic by

appear for

kinases

detergents calcium

(37). of

IL3

location

of

cells

involvement); known

rapid

the

nuclei it

exists

calcium

in

is

I

in

two

forms,

a

conserved

sequences substrate et

stimulated

correspond

criteria

for

only

al

have

detergent-insoluble

fibroblasts

not

KDa)

However,

(38).

Mahadevan of

(33

a significant

not

non-

released

chelation to

and

receptor

insoluble

lipocortin

antisera

of

does

(28)

EGF

to

33 by

the

various

topographical

pp33.

fulfills PP33 involved in growing

are

phosphorylation.

but

AX the

characteristically

however

by

phosphorylation

in (39),

KDa>

lipocortin

serine

protein

are

using

mediated

ligands

they

particularly

released

releasable

described KDa

they

studies

that

for

(31-35). (35

phospholipase including

kinases * Although

(36),

is

preliminary show

various

II

which

regulate

(29,30)

chelation

Lipocortin

one

to

certain

transduction.

signal before the effects

Thus,

of

withdrawal 527

on

intimately is


of IL3

it

necessary correlate changes

starvation

rapidity

protein

a

cell

present criterion well

in for with

metabolism,

BIOCHEMICAL

Vol. 164, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Low Trypsln

-45

-39 -27

c305Qc3060 I - IL-3 + IL-3 Pellets Fig. 6. 100,OOOg Control pp33 is pellets. stimulated

which

in

(15,

Effect of trypsln x 1 hr pellets pellets tC> were absent in both is present PP33 cells.

123

40,41j;

of

a

is

resting

This

a

into is

that but location

such

a

does

not

which

Unlike time

critical

kinetics, for

lost.

IL3

suggests

cells

control

a

division

unusual and

the

under

from

rapid

properties

IL3

as

entry

stimulate

dependent

cells

phase death

dynamic to

of

ensues

event.

the C20).

equilibrium pp33

be

IL3

a

has

the

candidate

molecule.

This project has been funded in part with from the Department of Health and Human Services contract with Program Resources Inc., and by a grant the Wellcome Trust to J.G.

Acknowledqments.

Federal

cell

biochemical

unusual

stimulate

a particular

before

controls

transport

growth which

cycle, at

glucose constitutive

ligands

arrest

withdrawn IL3

involve

becomes other

to

of pp33 in sonicetes. Arrow = pp33. and stimulated pellets from

cell

stimulate

phosphorylation

cell

when

(50 ug/ml) on stability from stimulated RB5 held on ice for 1 hr. control unstimulated in trypsin-treated

ligands

insufficient

cycle of

RB5

pp33

dependency have

and only

and

pp33;

SNS

funds

528

Vol. 164, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References 1. Carpenter G. (1987) Ann. Rev. Biochem. 56, 881-914. 2. Sibley D.R., Benovic J.L., Caron M.G. & Lefkowltz R.J. (1987) Cell 48. 913-922. 3. Hunter T. % Cooper J.A. (1981) Cell 24 741-752. 4. Davies R.J. (1988) J. Biolog. Chem. 263, 9462-9469. 5. Sherr C.J., Rettenmeier C.W., Sacca R., Roossel M.F., Look A. T. 8s Stanley E.R. (1985) Cell 41, 665-676. 6. Evans S., Rennick D. & Farrar W.L. (1986) Blood 68, 906913. 7. Koyasu S., Kasuga A., Tojo A., Miyajima T., Akiyama M., Urabe J., Schreurs K., Arai F., Takaku F. & Yahara I, (1987) EWBO J. 6, 3979-3984, . 8. Korla A.O., Schreurs J., Miyajima A. & Wang J.Y. (1988) Mol. Cell. Biol. 8. 2214-2218. 9. Park L., Friend D., Glllis S. % Urdal D.L. (1986) J. Biolog. Chem. 261, 205-210. 10. Nicola N. % Peterson L. (1986) J. Biolog. Chem. 261, 12384-12389. 11. Hapel A., Warren H.S. % Hume D.A. (1984) Blood 64, 786790. 12. LeGros G., Gillis S. % Watson J.D. (1985) J. Immunol. 135 4009-4019. 13. Birchenall-Sparks M., Farrar W.L., Rennick D., Killian P. % Ruscetti F.W. (1986) Science 233 455-458. 14. Garland J.M. (1988) Leuk. 2, 94-102. 15. Palacios R. % Garland J.M. (1984) Proc. Natnl. Acad. Sci. 81 1208-1211. 16. Garland J.M. (1984) Lymphokines 9, 154-201. 17. Moore K., Sullivan K., Tan E.M. % Prystowski M.B. (1987) 3. Biolog. Chem. 262, 8447-8450. 18. Lee M.G., Norbury C.J., Spurr N.K. % Nurse P. 11988> Nat. 333, 676-679. 19. Suzuki K. <1987) Trends Biochem. Sci. 12. 103-105. Martin S.R. &Thomas J.O. (1988) 20. Clark D.J., Hill C.S., EMBO J. 7, 69-75. 21. Alfageme C.R., Zweidler A., Mahowald A. & Cohen C.H. (1974) J. Biolog. Chem. 249, 3729-3736. Donahue M.J. (1984) J. Biolog. Chem. 22. % Masaracchia R.A. 259. 435-440. 23. Andres J.L., Johansen J.W. % Mailer J.L. (198'7) J. Biolog. Chem. 262, 14389-14376. Biolog. % Erikson R.L. C1987, J. 24. Blenis J., Kuo C.J. 14389-14393. Chem. 262, Hashimoto E., Mizuka K., Kondon H. % Yamamura 25. Sakanoe Y., (1987)Eur. J. Biochem. 168, 669-677. H. J.L. (1988) Ray L.B., Erikson E. % Maller 26. Sturrell T.W., Nat. 334, 715-718. Enzymol. 146, 36227. Siegmann M. 8s Thomas G. (1987) Meth. 369. 28. Touqui C., Rothhut B., Shaw A.M., Fradin A., Vargaftig B. % Russo-Marie F. <1986> Nat. 321, 177-179. Nat. 321. 81-84. L.K. (1986) 29. Pepinski R. % Sinclair EMBO J. 7. Marriott G. % Weber K. <1988) 30. Johnsson N., 2435-2442. Owens R.J. % Allan D., 31. Davies A., Wigglesworth N.M., Crumpton M.J. (1984) Biochem. J. 219, 301-308. (1984) Biochem. J. 219, 309Owens R.J. % Crumpton M.J. 32. 316. 33. Crompton M., Owens R.J., Totty N.F., Moss S.G. Waterfield cl9881 EMBO J. 7, 21-2'7. M.D. 8s Crumpton M.J. 529

Vol. 164, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Ceil 33, 767-779. 34. Greenberg M.E.% Edelman G.M. (lY83) Biochem. Biophys. Kes. 35. & Crumpton M.J. (1985) Davies A.A. comm. 128, 571-577. EMBO J. 3, 227-233. 36. Gerke V. 8s Weber K. (1984) % Burgess W.H. !I9871 J. 37. Haigler H.T., Schlaeffer D.D. Biolog. Chem. 262, 6921-6930. 38. Sheets E.E., Giugni T.D., Coates G.G., Schlaepfer D.D. % Halgler H.J. (1987) Biochem. 26, 1164-1172. 39. Mahadaven L., Heath J., Leichtfried F., Cumming D., Hirst E. % Foulkes G. (1988) Oncogene 2, 249-253. 40. Whetton A.D., Bazill G.W., % Dexter T.M. (19e4)EMBO J. 3. 409-411. 41. Garland J.M., Kinnaird A. % Elliott K. (1988) Leuk. 2, 476-480.

530