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Because dominant-negative. Ras17 has been shown to block the effects of PMA on pp42 MAP kinase phosphorylation, we assessed its effect on c-Jun ...
Vol.

4, 377-385,

May

1993

Cell

Multiple Signal Transduction c-Jun Protein Phosphorylation1

Christopher C. Franklin, Andrew S. Kraft2

Tino Unlap,

Victor

Adler,

and

Pathways

tion

Division of Hematology/Oncology, Birmingham, Birmingham, Alabama

University 35294

of Alabama

the

stimulate

esters) Abstract A variety of protein kinases, including pp42 and pp54 mitogen-activated protein (MAP) kinases, p342, and a partially purified protein kinase from 4$-phorbol 12myristate 13a-acetate (PMA)-treated U937 cells have been shown to phosphorylate the NH2-terminal activation domain of c-Jun in vitro. To investigate the role of pp42 MAP kinase in mediating c-Jun phosphorylation in vivo, we have treated U937 monocytic leukemia cells with a variety of pharmacological agents, including PMA, cycloheximide, AIF4, and okadaic acid. Although all of these agents stimulated c-Jun phosphorylation, cycloheximide and okadaic acid had no effect on pp42 MAP kinase phosphorylation, suggesting that MAP kinase activation was not necessary for c-Jun phosphorylation in vivo. Because dominant-negative Ras17 has been shown to block the effects of PMA on pp42 MAP kinase phosphorylation, we assessed its effect on c-Jun phosphorylation by cotransfection with a truncated c-Jun construct (c-Jun234). We found that cJun234 was expressed only in the cytosol and was inducibly phosphorylated with kinetics similar to those of endogenous nuclear c-Jun. Furthermore, we found that Ras17 had no effect on PMA-induced phosphorylation of c-Jun234. Because Ha-Ras requires isoprenylation for membrane binding, we examined the effect of the isoprenylation inhibitors lovastatin and perilhic acid on PMA-induced c-Jun phosphorylation. Pretreatment of U937 cells with these agents had no effect on PMA-induced c-Jun or pp42 MAP kinase phosphorylation. These data suggest that there are multiple pathways mediating c-Jun NH2-terminal phosphorylation in vivo, some of which are independent of MAP kinase activation, and that phorbol ester-mediated c-Jun and pp42 MAP kinase phosphorylation in U937 cells does not appear to be mediated by members of the Ras family. Introduction The c-Jun nuclear phosphoprotein of the AP-1 transcription factor

dimer can

or as a heterodimer bind

to AP-1

DNA

with sequences

is a major (1, 2). c-Jun

other and

component as a homo-

Jun or Fos proteins activate

cells contain

low levels of c-Jun c-jun gene is greatly by hormones (e.g., epidermal growth necrosis factor) and by compounds that

the

of

activity

the

of protein

(4, 5). Because

the

kinase

upstream

by the posttranslational

preexisting

protein

(6). c-Jun

appears

to be regulated

by both

terminal

phosphorylation.

boxy-terminal ity of c-Jun

to bind

to phenylalanine,

modification

transcriptional

amino-

to DNA

mimicking

of

activity

and

Phosphorylation

serine/threonine

phorbol

of the c-jun

and induction of cby protein synthetranscription of this

gene is regulated c-Jun

C (e.g.,

region

gene itself contains AP-1 sequences, jun expression is rapid and unaffected sis inhibitors, it is thought that the

carboxy-

of three

car-

residues inhibits the abil(7). Mutation of serine 243 a point mutation found in

v-Jun, blocks the phosphorylation of the other two residues, threonine 231 and serine 249, and enhances the DNA-binding activity of c-Jun (7). These three carboxyterminal sites can be phosphorylated by glycogen synthase kinase 3 (7) and CKII3 in vitro (8). Microinjection experiments using peptides that block CKII and the injection of CKII directly into cells suggest that the carboxy terminus of c-Jun is a substrate for CKII in vivo (8). c-Jun NH2-terminal phosphorylation also appears to play a role in mediating its transcriptional activity in vivo

(9-12). Phorbol stimulates the and

73 and

downstream

ester treatment phosphorylation

activates

the

of AP-1

of human of c-Jun

transcription

enhancer

leukemic cells on serines 63

of genes

elements

located

(9, 12). Studies

utilizing a fusion protein between the NH2 terminus of c-Jun and the DNA-binding domain of the yeast GAL4 protein have shown that the NH2-terminal 85 amino acids

of c-Jun

containing

these

two

phosphorylation

sites are

sufficient for transcriptional activation in vivo (1 2). Within this fusion protein, mutation of serines 63 and 73 to leucines abolishes PMA-induced transcriptional activity (12). Transfection of Ha-Ras, v-sis, v-src, or Raf-1 into fibroblasts also stimulates the phosphorylation of these serine residues (10, 1 1), suggesting that both oncogenes

and tumor promoters may modulate the same cascade of protein kinases. In contrast to these in vivo observations, in vitro transcriptional analyses suggest that NH2terminal phosphorylation does not play a significant role in regulating c-Jun transcriptional activity or DNA-binding activity (13). These discrepancies remain to be clarified. A number of protein kinases have been identified which are capable of phosphorylating the NH2 terminus of c-Jun. Both pp42 and ppS4 MAP kinases and p342

have been

shown

to phosphorylate

serines

63 and 73 of

transcrip-

The abbreviations used are: CKII, casein kinase II; PMA, 4l-phorbol 12myristale 1 3a-acetate; NGF, nerve growth factor; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel ehectrophoresis; kDa, kihodalton(s); GAP, GTPase-activating protein; CMV, cytomegahovirus; PBS, phosphatebuflered saline; BSA, bovine serum albumin. 3

Received 1/8/93; revised 2/1 1/93; accepted 2/1 7/93. ‘ This work was supported by National Cancer institute to A. S. K. and American Cancer Society Grant IRG-66-32 2 To whom requests for reprints should be addressed.

many

transcription

stimulated both factor and tumor

at

377

& Differentiation

Mediate

(3). Although

protein,

Growth

Grant CA42533 to C. C. F.

378

c-Jun Phosphorylation

through

Multiple

Pathways

bacterial c-Jun in vitro (9, 13). A protein kinase has recently been purified from PMA-treated U937 cells, using heparin-Sepharose and affinity chromatography, that is also capable of phosphorylating c-Jun and appears to be distinct from the previously characterized protein kinases (12, 14, 15). In addition to c-Jun, MAP kinase has also been shown to phosphorylate other transcription factors, including p62TcF (1 ) suggesting that this protein kinase may have a number of important nuclear targets. Although these protein kinases have been studied in vitro, the protein kinase(s) that phosphorylate c-Jun in vivo have not been identified. We have examined whether alternative pathways independent of pp42 MAP kinase may mediate the phosphorylation of c-Jun in human leukemic cells. Here, we demonstrate that the pharmacological agents cycloheximide, a protein synthesis inhibitor, and okadaic acid, a phosphatase inhibitor, induce c-Jun NH2-terminal phosphorylation but do not stimulate the phosphorylation of pp42 MAP kinase in U937 cells. ln contrast, PMA, a protein kinase C activator, and AIF4, an activator of heterotrimeric GTP-binding proteins, stimulate the phosphorylation of both proteins. Because it has been demonstrated that, in certain cell types, PMA-induced pp42 MAP kinase phosphorylation and activation can be blocked by expression of a dominant-inhibitory Ras protein (17, 18), Ras’17, we examined the effect of this protein on c-Jun phosphorylation in U937 cells. We found that expression of this Ras mutant had no effect on PMA-induced c-Jun phosphorylation in U937 cells. We also addressed the role of Ras by utilizing the isoprenylation inhibitors lovastatin and perillic acid to inhibit Ras processing and thus membrane association and biological activity (19-21). Although pretreatment of PC12 cells with these agents abolished PMA- and NGF-induced pp42 MAP kinase phosphorylation, which have been shown to be Ras dependent (1 7, 1 8, 22), they had no effect on PMA-induced pp42 MAP kinase or c-Jun phosphorylation

in

U937

cells.

These

PMA-induced phosphorylation independent of Ras in U937 biochemical pathways, some of pp42 MAP kinase or Ras activity, phosphorylation in these cells.

results

indicate

that

of pp42 MAP kinase is cells and that multiple which are distinct from exist that regulate c-Jun

Results To evaluate the role of pp42 MAP kinase as a putative cJun NH2-terminal protein kinase in vivo, we treated U937 human monocytic cells with several pharmacological agents known to modify various signal transduction pathways. The addition of either PMA or okadaic acid to U937 cells activates a macrophage-like differentiation which is accompanied by a rapid increase in c-Jun phosphorylation, induction of c-jun mRNA expression, and activation of AP-1-mediated increases in transcription (12, 23, 24). Cycloheximide treatment of U937 cells also increases c-Jun phosphorylation as well as causing the “superinduction” of c-jun and other immediate early genes by a variety of agents (5, 6, 25). We have found that U937 cells contain only the pp42 MAP kinase (ERK2), with no pp44 MAP kinase (ERK1) being detected by immunoblot analysis with an antibody that recognizes both MAP kinase proteins (data not shown). Phosphorylation of pp42 MAP kinase on both threonine 183 and

tyrosine 185 is necessary for the activation of MAP kinase activity (26). The mobility of the phosphorylated form of pp42 MAP kinase is retarded on SDS-polyacrylamide gels, yielding a doublet band. Likewise, we have previously shown that the ladder of c-Jun protein bands on SDS-polyacrylamide gels is due to the site-specific phosphorylation of serines 63 and 73 within the NH2-terminal activation domain of c-Jun (12). U937

cells

were

labeled

or 32P and treated

with

imide.

possible

Due

to the

with

PMA,

either

okadaic

[35S]methionine

acid,

temporal

or cyclohex-

differences

in pp42

MAP kinase and c-Jun phosphorylation in response to these stimuli, we investigated the time-dependent phosphorylation of these proteins. At the indicated times, cJun

and

pp42

MAP

kinase

were

immunoprecipitated

from whole cell denatured lysates. All three stimuli caused an increase in c-jun phosphorylation as judged by both an increased incorporation of 32P into c-jun and a retarded mobility of both 32P- and 35S-labeled c-Jun on SDS-PAGE (Fig. 1). However, only PMA stimulated an increase in pp42 MAP kinase phosphorylation. Cycloheximide and okadaic acid had no effect on either pp42 MAP kinase phosphorylation or mobility on SDS-PAGE (Fig. 1). The appearance of the retarded bands of protein on SDS-PAGE correlated well with the hyperphosphorylation

of both

c-Jun

and

pp42

MAP

our previous findings, which indicated in c-jun mobility is due to enhanced (12).

Although

the

apparent

sequential

kinase,

supporting

that the reduction phosphorylation phosphorylation

of pp42 MAP kinase and c-Jun in response to PMA treatment is consistent with pp42 MAP kinase-mediated c-jun phosphorylation, this hypothesis has yet to be verified. In any case, the ability of both okadaic acid and cycloheximide to induce c-jun phosphorylation in the absence of pp42 MAP kinase phosphorylation demonstrates the existence of a pp42 MAP kinase-independent pathway(s) of c-Jun phosphorylation in U937 cells. As a means of further examining the possible role of pp42 MAP kinase, we have attempted to alter the signal transduction pathway(s) that leads to pp42 MAP kinase phosphorylation and activation. Both oncogenic p21 Ras and activation of cellular p21 Ras have been shown to induce pp42 MAP kinase phosphorylation and kinase activity (17, 18, 27-29). In addition, the overexpression ofa dominant-inhibitory p21 Ras protein (Ras”7) inhibits the ability of insulin and platelet-derived growth factor in rat-i cells and of NGF in PC12 cells to stimulate the phosphorylation

of

pp42

MAP

kinase

(17,

18,

27-29).

These data suggest that Ras mediates tyrosine kinase receptor-induced MAP kinase activation. In PC12 cells, this mutant Ras protein also blocked the ability of PMA to stimulate the phosphorylation MAP kmnase; however, this effect appears to be cell type specific, as Rasnhl7 had no effect on PMA-induced phosphorylation of MAP kinase in rat-i fibroblasts (28). Because of the profound effect of this dominant-inhibitory Ras mutant on MAP kinase function, it was of interest to determine its effect on c-Jun phosphorylation. Attempts to establish a permanent U937 cell line stably expressing Ras17 were not successful, possibly due to the growth-inhibitory effects of this protein (30). To circumvent this problem, we took the approach of transiently

expressing

Rasbsr7

together

that is distinguishable from PAGE. We have previously

with

a c-Jun

construct

endogenous c-Jun on SDSassessed c-Jun posttransla-

Cell

PMA TIME

(mm)

0

5

15

O.A.

30

60

0

5

Growth

379

& Differentiation

CHX

15

30

60

0

5

15

30

60

mas*

#{149}#{149}#{149}

aaaS

S1S#{149}.

c-Jun 32

35s

*

32p

-*

-

-

-

-

.

-

MAPK

fig.

1. Time ourse of phosphorylation of c-lun and 1)1)42 MAP kinase (M.’PK( described in “Materials and Methods.” The cells were then treated with 0.2 M the indicated times, the cells were harvested, and whole cell denatured lysates and analyzed as described in “Materials and Methods.”

in U937 cells. PMA, 100 ng/ml were prepared.

tional modification ing a termination which encodes

microscopy. The majority of endogenous c-Jun was found in the nucleus, although a small portion was detected in the cytosolic fraction, possibly due to low levels of nuclear lysis (Fig. 2A). In both control and PMA-treated

by utilizing a c-Jun construct codon at amino acid 234 a protein lacking both the

contain(cJun2i4), basic and

leucine zipper regions (12). This truncated protein migrates separately from endogenous c-Jun on SDS-PAGE (29 kDa versus 39 kDa, respectively). c-Jun214 is hyperphosphorylated and shifted in mobility on SDS-PAGE in response to PMA treatment of U937 cells in a manner similar to that ofendogenous c-Jun (12). However, it was recently reported that the nuclear localization signal for c-Jun resides between amino acids 268 and 284 (31),

which

are deleted

in cJun2i4,

suggesting

that the cJun2i4

protein may be cytoplasmic. To determine the cellular location of c-Jun234, cell fractionation/immunoprecipitation studies were performed. U937 cells transiently transfected with cJun2i4 were labeled with fl5S]methionine and incubated in the absence or presence of PMA for 1 h. After a gentle hypotonic lysis procedure, the lysate was separated into crude nuclear and cytosolic fractions, and c-Jun was analyzed by immunoprecipitation. The nuclei isolated by this procedure were found to be greater than 95% intact as judged by phase contrast

cells,

U937

cells were labeled with ]#{176}S]me’thionine or ‘2P, as okadaic acid (0.4.), or 100 CM cycloheximide (CIIX(. At c-Ion and pp42 MAP kinase were immunoprecipitated

c-Jun234

plasmic

protein

fraction

the phosphorylation

(fig.

was 2A).

detected Furthermore,

of both

cytosolic

only

in the

PMA

cyto-

stimulated

c-Jun2t4

and

en-

dogenous nuclear c-Jun protein as judged by the retarded mobility of the proteins on SDS-PAGE (Fig. 2A; Ref. 12). As cell fractionation studies are complicated by nuclear lysis and leakage of proteins during the fractionation process, in situ c-jun immunofluorescence staining was performed. c-Jun immunofluorescence staining of Cos7 cells transiently transfected with either vector, c-jun234 or a holo-c-jun construct, was assessed using affinity-

purified peptide in cells

Whereas

antiserum (Oncogene transfected

raised against an NH2-terminal c-Jun Science). No staining was detected with

vector

alone

(Fig.

28,

Panel

3).

holo-c-Jun expression was restricted to the nucleus (Fig. 28, Panel 1 ), c-Jun254 staining was distributed throughout the cytosol (Fig. 2B, Panel 2). Thus, although cJun2i4 is expressed as a cytosolic protein in U937 cells,

380

c-Ion

Phosphorylation

through

Multiple

Pathways

A

B

46

kDa.

30

kDa-.

Cont

PMA

cy

cy nu

nu

#{149}

phase

immunofluorescence

1

contrast

4

c-Jun

,

#{149}

.,-

.

-5 .

Jt4c4i

tF’

2

k..

.,

,

5 -‘_‘4.4-

.‘r-

c-Jun

234

-

--

.

..

. ..

.

.‘..

,

.,

3

4

‘I,’.

6

vector

fig. 2 Cellular localization of c lun2” in U937 and Cos7 ( ills. .‘\. e’ll ir, tionation (ii U9 17 c ells expressing ( -lcjiY . U9 37 c e’lls 4 X 1O) ssem translected by elec troporation with ( -(un2’4. Eighteen h l)osttransl(’c lion, the ( ells were’ starved in methii)n(’-free media or 3() miii md then lal)i’l(’d with [ methionine for 4 h. During the last hour, the cells were split. and one-half were treated xx’ith 0.2 pM PMA. Cells were’ frac tionated into e vtosolic (cy) and nuc lear ( no ( fractions as des rihed in “Materials and Methods .“ F ndogenous -I un and ( - I ‘ pr(it(’ins were’ immunoprec ipitate(l from the fractions and analyzed by S[)S-PAGE. Unphosphorvl.3ted ( -lun2 and (‘n(li)g(’n(ius ( -lun migrate ii 29 and 19 kDa, re’sp(’( tixel . B, imniunotluores e’n( i’ staining (it Cos7 ( ells expressing c-Ion (it d -lun2 ‘. Cos7 (OIls seeded on glass ove’rslips were transiec ted with hololun, c-Ion2 , or expression vector alone by the ( alcium phosphate ( oprecipitalion proc(’dur(’. ( -lun immunotluiires ence staining was analyie’d 2 days posttransf(’cii(in using aitinity-purifiecl antiserum to an NH2-terminal C -(un peptide. as desc rihed in “Materials and Methods.” Right p,im’ls, the ( (irr(’sp(inding I)hase ontrast photographs.

un2

(‘oIl

Time

0

46

0.5

cvro

(hrs)

1

2

kDa-

4

6

-

MICROGRAMS I

I

Pulse-c

h,iso

,io,ilvsis

i ii

fi

PXIr\-indu

‘(f

(

-I on

111(1

(

( Lii)’

I)(1ii-

-

t)riiteiils

sx’en’

inlnluiliit)ri’

i1)it,lt(’d

md

,inalv,’il

by

Sl)’-P’\(E.

it is a substrate for PMA-inducible phosphorylation similar to that of endogenous nuclear c-Jun. These results cre not restricted to U937 cells, as similar results have also l)een observed in HepG2 cells (data not shown). To determine whether cytosolic c-lun24 was phosphorylated with similar kinetics to those of endogenous nuclear c-Jun, U937 cells were transfected with c-lun and pulse labeled with [SJmethionine. Cells were then chased with excess Linlabeled methionine in the presence of PMA, and the two c-Jun proteins were imniunoprecipitated at various times. The time course of c-Jun and c-Jun4 phosphorylation was identical, with maximal phos)hOryIation occurring between 30 mm and 1 h and decreasing thereafter (Fig. 3). PMA induces the activation of both cytosolic and nuclear pp42 MAP kinase in HeLa cells (32). We have recently isolated a protein kinase from PMA-treated U937 cells which is distinct from pp42 MAP kinase and is capable of phosphorylating serines 63 and 73 of c-Jun in vitro (14, 15). To determine whether c-Jun kinase activity is also present in an activated form in 1)0th the cytosol and nucleus of U937 cells, cell fractionation was performed in conjunction with in vitro cJun kinase assays. U937 cells were treated with PMA for 30 ruin, and crude nuclear and cytosolic fractions were isolated as described above. A fusion protein between the NH2-terminal 85 amino acids of c-Jun and glutathione S-translerase (GST-JUN) was used as a substrate for measuring c-Jun protein kinase activity as previously described (14, 15). PMA treatnient stimulated a protein kinase activity in both the cytosol and nucleus which was capal)Ie of phosphorylating the NH2 terminus of c-Jun (Fig. 4). Increasing amounts of added substrate yielded a concomitant increase in phosphor!ation. Although cJun2 is predominantly a cytosolic protein, it appears to be NH2-terminally phosphorylated in a manner identical to that of endogenous nuclear c-Jun. Thus, c-Jun2 4 appears to be a suitable substrate to analyze the effects of Ras” on PMA-induced c-Jun phosphorylation in U937 cells. To examine the role o Ras in PMA-induced c-Jun phosphorylation, U937 cells were cotransfected with c-

ii

Ii)

it

‘ri’

fli”i(

ii)

-I on

liii.itiiin

ifv

in

intl

iiiii-i,

ii iii

(

is

.15

55

,iss.ss

‘ri’

I)fi

iti

I

(,

‘iii

I -/1

10 50 100 OF GST-JUN

PPv1,\indu

iif

in

oil

.

luri

N f f-ti’rniin,il

LJ) I 7 ills. L (f 17 ((‘(Is SVi’t(’ tr(’at(’(l rudi’ iieeli’ar \t XL I ,iil vtiisiilu i)utlio(’(l “Nl,itorials md Methiicls.

1,01(1

si

ir,i( i it

phrvl.ltii)n. U9.3 7 ills sVi’t(’ i’l’ctropiiratocl svitli Iuo . md 24 h piisttransf(’ction. ( i’l(s sv(’r(’ ulS’ lal)el(’d svith nl(’thii iiluo’. Aft(’r 55 .ishiiig, the’ cells ss (‘re in iil),it(’(l in ( onipk’te nii’il a i iintaining (‘\( (‘s unlab’k’d nl(’thiiinin(’ md 0.2 xi PXIA. At the iiiiii ,it(’(l tinl(’s, ,lliclu(its sve’ri’ r(’no)v(’cI . .mnd svhiilo (‘II (l(’natur(’d lvsates \Si’t(’ Pri’t).ir’d. Both i len

-I. ((‘Ilul,ir

ic;.

Liii,isi’

kDa__#{216}I.

55

I i,’.

381

[)iff(’r(’nfialion

NUCL

10 50 100

30

Griisvth

l)Ot’\

\ I

(‘ii

for

(‘(‘0

,is

,i

in

glutathii ‘ul)str,it(’

itri

-lun pri)t(’in mc’ S-tr.insler.ise t 4 , 1 ‘i I. (



prot(’in

sx’ith (1.2 M PMA ) ( T( ) ) fr,utions /\liqu(its i)I e,ich

Lioasi’ ,i( .10(1 aniinii

tivity

using ,i

a

(Is S 89

Jury and the dominant-inhibitory Ras protein, Ras”’’. The ratio of Ras” to (-Jri11 DNA transfected was :1 to ensure that the cells expressing the c-Jun2 construct were also transfected with the Ras mutant. Immunopre(ipitation of Ras’ froni [ iSlnlethionine..labeled cells using an anti-Ras monoclonal antibody demonstrated that Ras” was expressed in great excess relative to that of endogenous Ras protein and was not modulated by the addition of PMA (Fig. 5B). Immunoprecipitation of cJun2 from Ras”-cotransfected cells indicated that transient expression of Ras’ did not block the PMAinduced phosphorylation of cJun2i (Fig 5A). These resuIts were not cell type specific for U937 cells, as identical results were obtained in HepG2 cells (data not shown). To further evaluate the role of Ras and other lowniolenilar-weight Ras-like GTP-binding I)roteins in mediating PMA-induced c-Jun phosphorylation, we utilized two pharmacological inhibitors of protein isoprenylation in an attenipt to block the biological function of Ras by inhibiting its ability to associate with the plasma membrane. It has been shown that several Ras proteins require isoprenylation for plasma membrane association and transforming activity (20). Although these compounds are known to inhibit Ras isoprenylation, they are also known to have other adverse effects, i.e. , inhibition of cholesterol biosynthesis and alterations in cell cycle progression (33). The effects of AIF.1 were also examined in U937 cells, as this compound is known to directly activate several heterotrimeric GTP-binding proteins which are thought to mediate its ability to stimulate pp42 MAP kinase activity (29, 34). To establish that inhibition of isoprenylation in fact inhibited Ras function, we investigated the effect of these inhibitors on PMAand NGFinduced p)42 MAP kinase phosphorylation in PCi 2 cells, which has previously been shown to be dependent on Ras lunction (17, 18). As has previously been reported, PMA and NGF stimulated the phosphorylation of pp42 MAP kinase in PC12 cells (Fig. 6A, Lanes 1-3). Pretreatment of PC12 cells with lovastatin and perillic acid, however, abolished pp42 MAP kinase phosphorylation in response to both PMA and NGF (Fig. 6A, Lanes 4-6). These results indicate that pharmacological inhibition o Ras processing can be effectively utilized to block Ras function. We utilized this phenomenon to address whether c-Jun phosphorylation in U937 cells was Ras

382

c-Ion

Phosphorylation

through

Multiple

Pathways

A

B Ras (Asnl7)

0 0

PMA

0

Ras (Asnl7)

PMA

0

PMA

0

0

Fig. S. Effect of dominant-inhibitory Ras (Ras’7( on PMA-induced c-Ion2” phosphorylation in U937 cells. U937 cells were transfected with c-Ion234 in the absence or presence ol excess Ras”7 (1:5 DNA ratio). Two days posttransfeclion, the cells were labeled with [35S]methionine, as described in “Materials and Methods.” The cells were then split, and one-half were treated with 0.2 LM PMA for 30 mm. lmmunoprecipitationof(A(c-

PMA

3044-

$‘

1

dependent. and

okadaic

234

U937

perillic

Ion2” and (B) Ras7 from whole cell denatured lysates was performed as described in Materials and Methods.”

21.5W

30

acid

acid,

cells and

5678

were

then

pretreated

treated

for

c-jun

and

or AIF4, and

with

lovastatin

30 mm

pp42

with

PMA,

MAP

kinase

ide did not stimulate the phosphorylation of pp42 MAP kinase. As threonine/tyrosine phosphorylation of pp42 MAP kinase is necessary for activation of protein kinase

phosphorylation were analyzed by immunoprecipitation (Fig. 6B). As previously shown in Fig. 1, PMA induced the phosphorylation of both proteins (Fig. 68, Lane 2), whereas okadaic acid stimulated c-Jun phosphorylation

activity (26, 35), these results suggest phorylation can occur in a pp42 MAP

in the absence

(36, 37), our results demonstrate that pp42 MAP kinase is not. The lack of an effect in response to okadaic acid is consistent with previous reports in PCi 2 cells, in which okadaic acid alone had no effect on pp42 MAP kinase but potentiated that observed in response to NGF (38). Only the time course of PMA-induced phosphorylation

of pp42

68, Lane 3). Similar

to

MAP

kinase

PMA,

AIF4

phosphorylation stimulated

(Fig.

both

pp42

MAP kinase and c-Jun phosphorylation (Fig. 68, Lane 4). Interestingly, inhibition of isoprenylation in U937 cells did not abolish the phosphorylation of either c-Jun or pp42

MAP

kinase

phosphorylation

in response

to any

of

the agonists tested, although the AIF4 response was reduced. Together, these results suggest that Ras does not play

a significant

role

in mediating

the

phosphorylation

of either pp42 MAP kinase or c-jun in U937 cells. Furthermore, it appears that PMA-induced pp42 MAP kinase phosphorylation occurs by both Ras-dependent and Rasindependent

mechanisms

in a cell

type-specific

manner.

of U937

cells

stimulates of c-Jun activation

the site-specific on serines of protein

63 and kinase

C by PMA, the exact signal transduction mechanism(s) by which PMA induces c-Jun phosphorylation remains unclear. A number of protein kinases have been identified which phosphorylate c-jun in vitro including pp42/ 44 and pp54 MAP kinases, p34(d(2 and a partially purified

enzyme from PMA-treated U937 cells which appears to be distinct from the others (9, 1 3- 1 5). However, because protein kinases promiscuously phosphorylate many subin vitro,

it is difficult

protein

kinases

to

identify

is the c-jun

which

of

these

NH2-terminal

pro-

tein kinase in vivo and which are modulated by phorbol esters. We utilized several very diverse pharmacological agents to examine the biochemical pathways responsible for c-jun phosphorylation in vivo. Whereas PMA, okadaic

acid,

cycloheximide,

phorylation

of

in U937

and AIF4 all stimulated cells,

okadaic

acid

and

c-Jun

phos-

cyclohexim-

in U937

pp42

cells.

Although

are activated

MAP

kinase

closely

Although directly address

plays

in mediating

a role

pp54

in response

phosphorylation. here did not

MAP

and

pp7O-

to cycloheximide

paralleled

that

the experiments whether pp42

PMA-induced

of

c-Jun

described MAP kinase

c-Jun

phospho-

rylation in vivo, they do suggest that there are other functional signaling pathways independent of pp42 MAP kinase that can mediate c-Jun phosphorylation in U937

both

NH2-terminal phosphorylation 73 (9, 1 2). Other than a direct

strates

manner

56 kinases

cells. We

Discussion PMA treatment

candidate

ent

that c-Jun phoskinase-independ-

have the

previously

basic

phosphorylated as those

cently Jun

and

on identical

mapped

reported resides

shown leucine

that zipper

serine

in endogenous

that the nuclear

in the

basic

protein (31), suggesting Using cell fractionation

leucine

c-jun234,

which

regions,

is inducibly

residues

(63 and

73)

It was

re-

c-Jun

localization zipper

(12).

signal region

lacks

for cof

the

that c-Jun254 might be cytosolic. and immunofluorescence stain-

ing, we have confirmed that this protein is localized to the cytosol. However, after phorbol ester treatment of U937 cells, this protein appears to be phosphorylated and dephosphorylated over an identical time course to that of endogenous nuclear c-Jun. Also, in vitro protein kinase assays demonstrate that both cytosolic and nuclear extracts contain a protein kinase activity capable of phosphorylating c-Jun. Therefore, PMA appears to stimulate both cytosolic and nuclear c-Jun NH2-terminal kinase activity in a similar manner. Serum and PMA treatment of HeLa cells has been reported to elicit a similar activation of both cytosolic and nuclear MAP kinase, which is accompanied by a translocation of MAP kinase

Cell

A .4-O

Lova.-#{248}

-04---

0

PMA

1

2

NGF

0

PMA

NGF

MAPK*II1$I

3

4

56

B 0 0

PMA

0-4---OA

AIF4

0

Lova.-* PMA

OA

AIF4

c-Jun

MAPK*

1

2345678

Fig. 6. Effect of the isoprenvlation inhibitors lovastatin and perillic acid on c-Ion and Pl42 MAP kinase (M,-\PK( phosphorylation. ‘1, effect of isoprenylation inhibitors on PMAand NGF-induced pp42 MAP kinase phosphorylation in PC12 cells. PC12 cells were’ Iett untreated or were pretreated for 20 h with 30 M lovastatin. Cells we’re then labeled for 4 h with [‘5Slmethionine in the absence’ (0) or presence of 30 pM lovastatin and 5 m perillic acid (Loca.). Cells were then treated with either 0.2 MM PMA or 100 ng/ml NGF for 15 mm, and pp42 MAP kinase was immonoprecipitated from whole cell denatured lysates as previously described. B, effect of isoprenylation inhibitors on c-Ion and pp42 MAP kinase phosphorylation in U937 cells. U937 cells were pretreated and 05 labeled exactly as described above’. Ce’lIs were then tre’ate’d with 0.2 pM PMA, 100 ng/ml okadai acid IO’\), or freshly prepare’d AIF4 as described in “Materials cipitated analyzed

and Methods.” from whole cell by SDS-PAGE.

c-Ion and denature’d

Pl42 MAP kinase were immunoprelysates as previously described and

activity from the cytosol to the nucleus (32). Attempts to address whether PMA induces the nuclear translocation of a recently identified c-Jun protein kinase were hindered by the complete lack of in vitro c-Jun kinase activity ofthis purified protein in cells nottreated with PMA (15). A dominant-inhibitory Ras mutant (Ras”7) has proved helpful in dissecting the cascade of events leading to the activation of pp42 MAP kinase by hormone receptor tyrosine kinases. Expression of this protein blocks the ability of NGF, insulin, or platelet-derived growth factor to stimulate the phosphorylation of pp42 MAP kinase in PC12 and rat-i cells (17, 18, 28). The role of Ras in mediating PMA-induced pp42 MAP kinase activity, however, appears to be cell type specific as Ras\s7 blocks the effects of PMA in PC12 cells but not in rat-i cells (17, 18, 28). A similar inhibitory effect has been reported in

Growth

& Differentiation

NIH 3T3 cells overexpressing GAP, which also acts to negatively regulate Ras activity (29). We attempted to evaluate PMA- and NGF-induced pp42 MAP kinase and c-Jun phosphorylation in the above-mentioned Ras” PC12 cells, but even under stringent control of the culture conditions, the negative regulatory effects of Ras” could be detected under uninduced conditions.4 Attempts to establish a U937 cell line which contained such an inducible mutant Ras were unsuccessful, suggesting that this gene product is highly toxic even at low levels in U937 cells (30). Similar difficulties have been overcome in NIH 3T3 cells by prior transformation with vRaf, which overcomes the growth-inhibitory effects of Ras””7 (22). This approach was not feasible for our studies, as v-Raf has been shown to activate MAP kinases, albeit indirectly (39-41). We therefore decided to examine the effect of transient transfection of Ras on c-Jun phosphorylation in U937 cells. To examine the ability of RasA57 to modulate the activity of phorbol esters, this protein was cotransfected with c-Jun214. Whereas Ras7 protein was expressed at levels far exceeding those of endogenous Ras protein, it appeared to have no effect on PMA-mediated cJun2i4 phosphorylation in U937 cells. As mentioned above, it is possible that the effects of Ras”’7 are cell type specific, since it blocked phorbol ester-induced effects in PC12 cells and not in Rat-i cells. However, we did not see any effect of this mutant on PMA-induced phosphorylation of c-jun24 in HepG2 cells (data not shown). We cannot exclude the possibility that nuclear (endogenous) and cytosolic (c-Jun2#{176}) c-Jun are phosphorylated by distinct mechanisms due to their differential compartmentalization. Many reports suggest that the Ras proteins may play a role in phorbol ester-mediated growth responses. Overexpression of GAP in mouse NIH 3T3 cells blocked the ability of PMA to stimulate MAP kinase phosphorylation (29). PMA treatment of T-cells has been shown to inhibit GAP and induce the formation of a more active GTPbound form of Ras (42). To evaluate the role of Ras proteins in the regulation of c-Jun phosphorylation, we treated U937 cells with perillic acid and lovastatin, both of which inhibit the isoprenylation of low-molecularweight GTP-binding proteins (19, 21). Both Harvey and Kirsten Ras proteins have been shown to require isoprenylation to associate with the cell membrane (20). Using PC12 cells, we have demonstrated that pharmacological inhibition of Ras isoprenylation is a viable means of inhibiting Ras-mediated cellular events. However, treatment of U937 cells with these compounds did not affect PMA-induced phosphorylation of either pp42 MAP kinase or c-Jun. These results indicate that c-Jun phosphorylation in U937 cells is independent of Ras activity and furthermore provide evidence that Ras-dependent and Ras-independent mechanisms mediate pp42 MAP kinase phosphorylation in a cell type-specific manner. It therefore appears that multiple pathways are involved in mediating c-Jun phosphorylation in U937 cells. Both genetic (Ras7) and pharmacological (isoprenylation inhibitors) manipulation of the Ras signaling path-

4

C. C.

Franklin

and

A.

S. Kraft,

unpublished

ol)servations.

383

384

c-Jun

Phosphorylation

ways

indicates

phorylation We cannot

through

that

Multiple

phorbol

Pathways

ester-mediated

in these cells is not mediated rule out the possibility that

c-Jun

phos-

by Ras proteins. other pathways

may predominate in other mouse or human cell systems that are not of hematopoietic origin. Although the exact functional role of c-Jun NH2-terminal phosphorylation in

transcriptional regulation remains to be more fully evaluated, phosphorylation of this protein in response to a variety of stimuli serves as a means for evaluating the cascade of events that regulate nuclear protein phosphorylation.

by immunoprecipitation with a v-H-ras monoclonal antibody (clone Yi 3-238; Oncogene Science), and immune complexes were collected with protein-G-Sepharose following the manufacturer’s instructions.

Cell nuclear

cells

Fractionation

and

and

fractions

cytosolic

in hypotonic

lmmunofluorescence.

Nonidet

were

insoluble

by

P-40 lysis buffer

pH 7.5-iO mM NaCI-3 mi MgCI2-0.5 ylsulfonyl fluoride-0.05% Nonidet P-40) Following a low-speed spin (500 x g), was collected and recentrifuged for iS

g to remove

Crude

obtained

material

lysing

(iO mt’i Tris,

mti phenylmethon ice for 5 mm. the supernatant mm at i2,000 x

and was designated

the

Materials and Methods Cell Culture. U937 cells were cultured in Dulbecco’s modified Eagle’s medium containing iO% iron-supplemented, heat-inactivated bovine calf serum, 100 units/ ml penicillin, and 100 zg/ml streptomycin in a humidified atmosphere of 5% C02-95% air. PCi2 cells were obtamed from the American Type Culture Collection and were maintained in monolayers in Dulbecco’s modified Eagle’s medium supplemented with iO% heat-mactivated horse serum, 5% fetal calf serum, 4 mr’i glutammne, and antibiotics. Cells were treated by direct addition of either 200 flM PMA, 100 ng/mI NGF (GIBCO), 100 M cycloheximide, iOO ng/ml okadaic acid (GIBCO), or fluoroaluminate prepared fresh daily by mixing 300 mrs’i NaF with 1 mM AICI3 at a ratio of 10:1 (final concentration, 30 mM NaF and iO LM AlCl3) (34). Lovastatin (the kind gift of Dr. A. Alberts, Merck, Sharp and Dohme) and perillic acid (Janssen Chimica) were used at 30 zM and 5 mrvt, respectively. Plasmids and Transfedions. The c-jun234 construct has been described previously (12). The dominant-inhibitory Ras expression vector, CMV-Ras”7, was prepared as follows. Plasmid pXVR (the kind gift of Dr. L. Feig) was digested with BamHl and Xbal, and a 1.i-kilobase frag-

cytosolic fraction. The crude nuclear pellet was washed immediately with lysis buffer. Nuclei were found to be greater than 95% intact as judged by phase contrast microscopy. Nuclei were lysed by boiling for S mm in denaturing lysis buffer (50 mt Tris, pH 8.0-0.5% SDS-5 mM dithiothreitol-0.5 mi phenylmethylsulfonyl fluoride),

luted

i:iO

in i%

ment

with

PBS,

blocked

of the

v-Ha-ras

gene

was

excised

and

cloned

into

pBluescript SK (Stratagene). The insert was then excised with BamHl and Notl and cloned into the pCEP4-CMV expression vector (Invitrogen). U937 cells were transfected by electroporation as previously described (i2). Approximately 2-3 x i07 cells were transfected in 0.5 ml PBS with 30-50 jzg DNA at a setting of 250 V and 250 jtfarads. When c-Jun234 and Ras17 were cotransfected, the amounts of DNA used were 10 and 50 zg, respectively. Cell Labeling and lmmunoprecipitation. U937 cells were labeled as previously described (12). PC12 cells were preincubated for 30 mm in minimal essential medium containing 0.5% dialyzed bovine calf serum and lacking either methionine or phosphate. Cells were Iabeled for 2-6 h in the same media containing either 0.i mCi/mI Trans35S-label or 0.5-i mCi/mi 32P, respectively. After

cell

treatment,

whole

cell

denatured

lysates

were

prepared as previously described (12). All lysates were precleared overnight with Protein A-Sepharose (Pharmacia). c-jun. c-Jun234, and pp42 MAP kinase were immunoprecipitated with c-jun-specific polyclonal rabbit antisera raised against a pGEX-c-jun fusion protein (1:S00 dilution) or pp42-specific antisera (1 :500 dilution; the gift of Dr. J. Ferrell, Stanford University, Palo Alto, CA), respectively.

Protein by

iO%

Immune

A-Sepharose, SDS-PAGE.

complexes

were

collected

with

washed extensively, and analyzed Expression of Ras#{176}7was analyzed

followed

by a 5-fold

dilution

with

radioimmunoprecipi-

tation assay buffer lacking SDS. c-Jun was immunoprecipitated from the nuclear and cytosolic fractions as described above. c-Jun immunofluorescence staining was performed in Cos7 cells transfected with CMV-holo-c-Jun, CMV-cJun234, or pCEP4 expression vector alone. Cells were

plated on glass coverslips and transfected with 5 jzg of plasmid DNA by the calcium phosphate coprecipitation method. Two days posttransfection, cells were washed with

PBS and

fixed

with

for 45 mm at room

PBS containing

temperature.

3% formaldehyde

Coverslips

were

washed

with PBS and incubated in ice-cold acetone for 20 mm. After rinsing, the cells were blocked in i% BSA-PBS for 20 mm at room temperature. Coverslips were then incubated for 30 mm at 37#{176}Cwith affinity-purified c-Jun polyclonal antibody (c-Jun Ab-2; Oncogene Science) di-

37#{176}C for

BSA-PBS. in

1 h with

Coverslips

1%

goat

were

then

and

incubated

BSA-PBS,

anti-rabbit

washed

fluorescein

cyanate (Southern Biotech, Birmingham, in 1% BSA-PBS. Coverslips were washed ered with Hoechst dye, and mounted fluorescence microscopy was performed.

at

isothio-

AL) diluted with PBS, on slides,

i :50 coyand

Acknowledgments The authors thank Dr. I. Ferrell for the kind gift of the pp42 MAP kinase antisera, Dr. L. Feig for the dominant-negative Ras expression vector, Dr. S. Halegoua for the PC12 cell line containing the dominant-negative Ras mutant, Dr. A. Alberts for the lovastatin, Dr. A. Morris for helpful discossions, and F. Wagner for technical assistance.

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