Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells

Vol. 189, No. 1, 1992 November 30, 1992

BIOCHEMICAL

IMMIJNOCYTOCHKMICAL EXPRESSION

AND BIOPHYSICAL

AND LOCALIZATION

AORTIC ENDOTHELIAL Oscar

R. Resales*,

Carlos

of *Medicine

E.

October

1,

C IN BOVINE

CELLS1

Sumpio+ Yale

and *Surgery, New Haven,

Received

OF PROTEIN KINASE

Isales*,MichaelNathanson*,

and Bauer Departments

RESEARCH COMMUNICATIONS Pages 40-46

University

School

of Medicine,

CT 06510

1992

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cytosolic fraction (66%)in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the a and B PKC isoforms. y PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC a immunofluorescence redistributed to the perinuclear region whereas PKC B staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation. 0 1992 RcademlcPress, Inc.

Protein protein of

kinase

kinases

which

many cell

Cloning that

systems

studies encode

have

of

reported;

additional will

thought

to

calcium,

both

to

occur

a group

a variety

of

demonstrated of

in

to

of the following

a variety

of of

cell expression

the

activation cell

including of

the

a family exhibit

in

the

and drugs

(1).

of closely

related

genes

(2).

future.

cultured

of

40

of

y)

were (2)

PKC is

diacylglycerol

hydrolysis

and

of membrane

and particulate

fractions.

in membrane-associated has been However,

in BAEC have

1Supported by grants to B.E.S. from the NIH (HL40305, Administration. O.R.R. was an AHA Research Fellow,

and

been isolated

"translocation" BAEC (3).

B,

Activation

action

cytosolic This

tissue-specific (a,

subsequently

by signal-induced

in both

response

factors

genes

synergistic

PKC isoenzymes

0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

transduction

distinct

different

of PKC is an increase

stimulation

types

signal

growth

a and 0 have

can be generated

PKC can be measured

An indication

which

(a,

and phospholipid-dependent

in the

three

be discovered

response

calcium-

PKC is

isoenzymes

cDNA clones

of which

of

hormones,

that

Initially,

probably

phospholipids.

patterns

to be involved

expression.

others

activity

is

appears

a number

patterns and

C (PKC)

not

HL47345), Connecticut

the been

PKC noted

in

individual described.

and the Veterans Affiliate.

Vol.

189,

No.

Using analysis

both

with

intracellular

BIOCHEMICAL

1, 1992

PKC activity

confocal

demonstrated

a predominant

isoform

of

13-acetate

(TPA),

Brief led

By immunofluorescent of

translocation

cytosolic

could

PKC.

be observed stimulation

to translocation staining

stimulation and increase

with

the

PKC activity against

the with of

effect

in

confluent R and of the

the BAEC

antibody with

immunocytochemistry of

calf

BAEC. y

serum By

isoforms

PKC,

a and R isoforms directed

on the

utilizing of

we

of PKC

against

to a particulate

a dense

perinuclear

PKC R remained was blocked

COMMUNICATIONS

the

y

12-O-tetradecanoylphorbol

of PKC activity

TPA whereas

and

a,

localization

PKC a displayed in activitv

RESEARCH

assays

we examined

directed

in BAEC. No staining

10 min

of

antibodies

BIOPHYSICAL

translocation

microscopy,

distribution

monoclonal

AND

in

bv caluhostin

fraction. pattern

the

cytosol.

after PKC

C.

METHODS Exoerimental Protocol BAEC were obtained by gentle scraping of the intima of calf thoracic aorta and maintained in DMEM F12 supplemented with 10% heat inactivated calf serum, 5 pg/ml deoxycytidine, 5 pg/ml thymidine, 1% penicillin-streptomycin and fungizone. BAEC were grown to confluence in 35 mm polystyrene wells and maintained in 10% calf serum for 24 hrs prior to experimentation (FBS) or serum-deprived for 24 hrs (SDM). BAEC were then treated with either the vehicle (0.1% DMSO) for 10 min or with 100 nM TPA dissolved in 0.1% DMSO for 10 min. In other studies, 0.05 pM calphostin C (Kamiya, Mountain View, CA) was activated by light (4) and added 30 min prior to the addition of TPA. Experiments were performed in quadruplicate. Cvtosol and Membrane Fractionation BAEC were washed twice with cold PBS and with buffer A (20 mM tris-HCl, pH 7.5, containing 2 mM EDTA, 0.5 mM EGTA, 2 mM PMSF, 25 pg/ml leupeptin, and 0.33 M sucrose) and scraped from the wells into 4 ml of buffer A. The suspension was homogenized and centrifuged at1500 g for 15 min. Completeness of cell disruption was confirmed by light microscopic evaluation of an aliquot of the suspension. The supernatant (cytosolic fraction) was saved and the pellet (membrane fraction) was washed in 5 ml of buffer B (20 mM tris-HCL, pH 7.5, containing 2 mM EDTA, 0.5 mM EGTA, 2 mM PMSF), centrifuged and resuspended in 4 ml of buffer B. Protein determinations were performed by the method of Bradford (5). To solubilize PKC associated with the membrane fraction, 1% NP-40 in buffer B was added and then centrifuged to obtain detergent-solubilized PKC from the membrane fraction. Both soluble and detergent-solubilized particulate fractions were applied to a DE-52 cellulose (Whatman) columnpolypropylene columns (Macalester Bicknell, New Haven, CT), equilibratedwithbuffer consisting of 20 mM Tris, 0.5 mM EDTA, 0.5 mM EGTA, and 5 mM R-mercaptoethanol (pH 7.5). After the columns were washed, PKC was eluted with equilibration buffer containing 150 mM NaCl. Protein Kinase C Assay Calciumand phospholipid dependent PKC activities were measured by determining 32P transferred from [Y-~~P]-ATP (specific activity lo-25 Ci/mmol, New England Nuclear, Boston, MA) to histone III-S as described by Kraft et al (6). In brief, l-3 pg of protein from the membrane or cytosolic fractions of BAEC were assayed in a reaction mixture (200 pl) containing 20 mM Tris-HCl (pH 7.5), 10 mM MgC12, 5 &ml 1,2-diolein, 200 pg/ml histone III-S, in the absence or presence of 0.5 mM CaC12 and 25 pg/ml phosphatidylserine. The reaction was initiated by addition of 10 pl of 0.4 mM [Y-~~P]ATP (250-500 cpm/pmol), for 10 min at 30°C. Twenty five pl aliquots were transferred to 1.5 cm2 Whatman P-81phosphocellulose the paper washed three times in 75 mM phosphoric acid, and 32P determined paper, by standard liquid scintillation techniques. PKC activity was determined by subtracting the amount of 32P incorporated into histone in the absence of calcium and phospholipid from the amount of 32P incorporated into histone in the presence of calcium and phospholipid and was expressed as nmoles of 32P incorporated/mg of protein/min. 41

Vol.

189, No. 1, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Immunocvtofluorescence of PKC Isoenzvmes Immunocytochemistry of PKC isoenzymes was examinedby slight modifications of previously reported methods (7,8). We used monoclonal antibodies directed against rabbit brain-PKC that react specifically with the a (MC-3a), B (MC-2a) and y (MC-la) subspecies of PKC (Seikagaku Kogyo Co., Ltd, Tokyo, Japan). The specificity of these antibodies for the different PKC-isoenzymes has been assessed by immunocytochemical analysis of the rabbit cerebellum (7). BAEC were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 30 min and incubated with 50 mM ammonium chloride for 15 min. After incubation with 1% bovine serum albumin in phosphate buffer solution (BSA/PBS) for 60 min, the preparation was incubated for 2 hrs at'room temperature with the PKC antibodies diluted in 1% BSA/PBS (1:20), washed three times with 1% BSA/PBS and subsequently exposed to the rhodamine-conjugated secondary antibody (l:lOO, 1% BSA/PBS) for 60 min. The preparation was then mounted with 50% glycerol and observed with a 40x or 63x objective on a Zeiss Axiovert microscope using a BioRad MRC-600 Confocal Imaging System. Aperture, gain and black level for imaging acquisition were maintained constant. No background or autofluorescence was visible at the chosen settings. Statistical Analvsis Results are expressed as mean+ standard deviation. The effect of different incubation media and TPA on PKC activity was analyzed by the unpaired Students' t test. Differences were considered significant at p < 0.05 level.

RESULTS PKC Activitv

in Cvtosolic

Total

PKC

nmoles/mg/min. fraction BAEC

to

100

of

calphostin

the

C prior

Total nmoles/mg/min, contrast activity of

arising

in

of

a reduction to

in

in cytosolic

a significant

(p
with

Localization Figure subspecies

of

cytosol. to

activity

PKC Isoenzvmes localized

that

from

the

in

the

30 min with drop

the

FBS

total

0.05

in total activity

the

fraction 1.6

BAEC

1.3f0.2

of

the

PKC

(69%),

with

only

31%

10 minutes

fraction

Pre-treatment PKC activity

In

majority

nmole/mg/min

the cytosolic

was

SDM BAEC (p
that

particulate

total

to

fraction.

100 nM TPA for

(3%). in

contributions

2 demonstrates were

the

60% of

unstimulated

to

drop

in a significant

of

1.520.2

fraction for

confluent

for

to

a commensurate

particulate

0.9kO.l

exposure

increased

with

1 demonstrates

decrease

equal

resulted (p-0.001)

PKC activity

PKC activity

After

particulate

in the particulate

the

total

fraction.

was

was

was in the membrane

to 9%. Pre-treatment

than

Figure

SDM BAEC activity

the

there

unstimulated

was higher

from

rise

translocation

Both

activity

in FBS BAEC was located

activity

of

while

fraction

BAEC

PKC activity

and 40% in the

SDM cells,

significant

led

contribution 91%,

nmoles/mg/min

which

to

total

to

total

cytosolic

to TPA stimulation,

fraction PKC

the

The

cytosolic

to 0.5fO.l

cytosolic

in

of

confluent

34% of the

10 min,

increased

PKC activity the

for

Fractions

unstimulated that

activity

(p-0.005).

activity

in

the

nM TPA

contribution pM

in

1 shows

66% of

nmoles/mg/min total

activity

Figure

and

and Particulate

(97%) with

0.05

to

0.650.1

also

caused

a

(p
with

associated

with

pM calphostin

C

nmoles/mg/min

and particulate

fractions.

in BAEC SDM BAEC expressed predominantly 42

in the

the

a and 13 isoforms

cytosol,

extending

of PKC. from

the

Vol.

189,

No.

1, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

2

Fizure

COMMUNICATIONS

*

1.

Effect of TPA on intracellular partitioning of protein kinase C. Confluent BAEC in either serum-derived media (SDM) or in media containing 10% FBS were exposed to either 0.1% DMSO (Basal) or 100 nM 12-O-tetradecanoylphorbol13-acetate (TPA) in 0.1% DMSO for 10 minutes. In some experiments 0.05 pM calphostin c (CC) was added for 30 min prior to stimulation with TPA. After homogenization and high speed centrifugation, PKC activities were measured in the cytosolic (clear bars) andmembrane (hatchedbars) fractions (nmoles/mgprotein/min, mean+ SD). *p
plasmamembrane observed. y or

to the nuclear

No staining

in

membrane

was observed

preparations

that

did

with

no significant

utilizing

not

the

include

intranuclear

antibody

primary

directed

staining against

antibodies

to

the

PKC

a or

R

isoforms. Incubation

of

redistribution stain

of

with

cytosol

and

although

r-24 TPA

remaining

PKC B. PKC a fluorescence

some intranuclear around

Pre-incubation

cytoplasmic of

the

plasma

0.05

serum,

membrane.

were

of both obtained

although

10

and less

significant intense

faint

The

fluorescent

revealed to

mild

43

of of

a marked

changes in

the

in

the

perinuclear

periphery

pattern

of of

perinuclear

TPA stimulation

experiments

translocation

caused

in the

a and B isoforms the

min

was most

C prior when

the

for

and very

cytosolic,

pM calphostin

staining

results calf

staining,

predominantly with

Similar presence

100

in PKC a immunofluorescence

pattern

region,

diffuse

SDM BAEC with

the

PKC B,

staining. resulted

in

PKC. were

performed

in the

PKC a immunofluorescence

Vol.

189, No. 1, 1992

BIOCHEMICAL

Basal

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

100 nM TPA

Calphostin

C

PKC a

PKC p

Figure

2,

Confocal fluorescence microscopy ofproteinkinase C 1ocalizationinBAEC. Images were focussed at the middle portion of the nucleus. (Left panel) BAEC were exposed to 0.1% DMSO for 10 min, fixed, and incubated with antibodies against PKC e, B, or y for 2 hrs, and subsequently exposed to the rhodamine-labelled anti-mouse IgG for 60 minutes (n-6). BAEC incubated without primary antibody or with PKC y failed to display immunofluorescence. (Middle panel) Exposure of BAEC to 100 nM TPA in 0.1% DMSO for 10 min, resulted in a shift of PKC a to the perinuclear region, whereas the fluorescent pattern of PKC B remained predominantly cytosolic (n-6). (Right panel) 0.05 PM calphostin C was added to BAEC for 30 min prior to exposure to 100 nM TPA in 0.1% DMSO for 10 min. There was diffuse cytoplasmic staining

of

both

was not

as striking

already

localized

a and

B isoforms

since

under

to

the

of

basal

PKC.

conditions

particulate

the majority

component

(Figure

of PKC activity

was

1).

DISCUSSION

Immunohistochemistrywithconfocal advantages

over

reconstruction accurate deprived

EC, to

antibody

which

PKC a and

The

Similarly,

under to

hence

we

of the

e and ( isozymes

of

PKC I3 cross-reacts cannot

current of

is

distinguish

primary with

(9).

In resting

of

expression

of

and a more

expressed

serumand

is

BAEC incubated

with

and

antibody

both

between

unavailability

PKC, the

fixing

similarly in

resolution

upon

distribution

was observed

a variety

distinct

includinghigher

distortion

I3 immunofluorescence No staining

antibody

and

microscopy

may suffer

or fluorescence

cytosol.

in view

8,

of stain

PKC y even

subspecies, the

fluorescence

the

to

dilutions.

still

conventional of structures

assessment

localized

fluorescencemicroscopyoffers

secondary the

131 and

these

antibodies these

two

82 PKC

isoenzymes.

directed

subspecies

our

against in BAEC is

unknown. The ratio

according

to cell

of the type,

membrane

to soluble

degree

of confluence 44

PKC activity

in resting

and differentiation

cells

varies

and incubation

Vol.

189,

No.

media.

In

about in

this

study,

This

observation

0.5.

the

BIOCHEMICAL

1, 1992

same cells,

cytosol. (10)

cytosol

or

ratio.

activity

For

maintained

of change

to membrane

cytosol

Recent

reports

results

not

fraction with

only

other in

cell

binding

to

of

PKC isoenzymes

activation

of

this

in

a higher

that

the

and NIH-3T3

In contrast,

cells

membrane

67% of the cells

percentage

Nonetheless,

our

to

total

PKC

incubated

in

in confluent

BAEC

TPA for

effects

10 minutes

perinuclear

staining

stained

the for

different

cell

perhaps

reflecting include

both

(16)

reported

that

dibutyrate,

membrane. NIH

Leach

studies,

systems of

HL60

(17)

using

et

types

another. with

in BAEC.

100 nM

mostly

PKC ct to

the

cytosolic

and

examined

al

(10)

also 45

studies

have

as intact

cells.

with

bryostatin

1,

the

of a PKC-like

increase showed

been diverse,

against pattern,

Hocevar

13-acetate in nuclear increased

et

al

phorbol

activity

to the

localization

PKC a. while

used

and not

immunocytochemical

directed

several

protocols

as well

cytoplasmic

a specific

soughtin

and experimental

withphorbol12-myristate

of PKC with Halsey

cells

antibodies

a diffuse

of cells

prevent

treatment of

been

of these

and translocation

et al in

relocation

PKC localizationhas of cell

the activation

3T3 cells

Treatment

range

treatment

PKC 01 localized

in a redistribution

wide

for

not

that

PKC R remained

The results

reconstituted

caused

PKC in

nuclear

and tissues. the

while

but

expression

intense

the

or may mask the

site

suggest

One of to assay

region.

or against

types

which

regions

perinuclear

one

studies

and

the

completely

compartments

of PKC isotype

an apparent

throughout (1).

may not

in

(6,17). how

effects

at

also

may explain

fractionation

cellular

BAEC. of PKC

but

elements

Fixation

fed

structures

of proteins

antibody

on patterns

caused

(12,13),

biological

putative

serum activation

and nuclear

of subcellular

monoclonal

that

lipids

in a

was detected

and the

cellular

and diverse

and intranuclear

Evidence

membrane

than

of PKC activity

PKC activity

demonstrated

(14,15)

EC, phorbol

rather

Translocation

in phosphorylation

other

one

including

PKC activity

deprived

different

immunofluorescent

TPA has different

have

bywesternblot.

from by

types,

>91% of

serum

is the absence

or PKC isozymes recognized

that

the

to

of multiple

loss of PKC isozymes eptiope

cell

(3,12).

plasma

results

of our study

PKC activity

these

evidence

expressed

endothelial

of

elements

enzyme

and regulation

limitations

to

reported

inmany

systems

Translocation

unstained.

BAEC was

BALB/c-3T3

display

a similar

such

in both

cytoskeletal

cells,

(3)

mainly in

EC (11).

arterial

PKC activity

occurred,

to

of

is

media

al

deprived

immunocytochemical

reported

a redistribution

translocation

nuclear

et

studies

in total

particulate

faintly

our

aortic

We found

EC causes

from

cell

been

pulmonary

from previous

treatment

the

with

porcine

Myers

associated.

quantitative in

confluent-serum

in serum-rich

bovine

COMMUNICATIONS

in 10% FBS.

As expected ester

have

quiescent

instance,

10% FBS was membrane

in

RESEARCH

PKC immunofluorescence

incubated

in confluent

ratio

ratios

sparse,

growing

BIOPHYSICAL

is in agreement

activity

and in

actively

activity

in which

Similar

cells

the

AND

the

In

control

nuclei

were

(PMA) resulted PKC. In contrast

perinuclear

staining

Vol.

in

189,

No.

PMA-treated

activity

BIOCHEMICAL

1, 1992

3T3-Ll

cells

was associated

using

only

with

AND

BIOPHYSICAL

a polyclonal nonnuclear

RESEARCH

antibody

COMMUNICATIONS

to

membranes

PKG. However,

and not

with

the

PKC

nuclear

fraction. affect and

The

exact

role

the

transcription

interferon

proteins

PKC in

been

and only

systems,

envelope.

nucleus

PMA-stimulated reported,

II

some of Thus,

these

the

is

not

including

yet

lamin

(19). proteins,

B (16),

such

Phorbol

plasminogen of

Most of these

physiological

known.

c-fos,

phosphorylation

including

and DNA topoisomerase

nuclear

the

of many genes,

(17).

has

proteins

of

studies

as lamin

PKC nuclear

activator

a variety histones used B,

are

esters

of

nuclear

(18),

matrix

reconstitution located

substrates

in

have

not

the been

identified. In that

BAEC express

treatment

of

membrane areas.

the

present

PKC a and

EC results

fractions, Further

isoenzymes insights

in

summary,

in to the

in

the

multiple

we provide

I3 isotypes,

but

immunocytochemical not

y.

translocation

of

PKC activity

a and 13 isoforms

of

PKC appear

understanding response

study

of

to

different

regulatory

the

intracellular agonists effects

phorbol

from to

location and

of

Although

antagonists

evidence

the

ester

cytosol

shift

to

different

of

the

different

to

may contribute

PKC.

REFERENCES

1. Nishizuka, Y.. (1988) Nature 334: 661-665. 2. Blackshear P.J. (1988) Am. J. Med. Sci. 296: 231-240. 3. Myers, C.L., J.S. Laze, B.R. Pitt. (1989) Am. J. Physiol. 257: L253-L258. 4. Bruns R.F., Miller F.D., Merriman R.L., Howbert J.J., Heath W.F., Kobayashi E ., Takahashi I., Tamaoki T., Nakano H. (1991) Biochem Biophy Res Comm 176:288293. 5. Bradford, M.M. (1976) Anal. Biochem. 72:248-254. 6. Kraft, A.S., W.B. Anderson, H.L. Cooper, J.J. Sando. (1982) J. Biol. Chem. 257: 13193-13196. 7. Hidaka, H., T. Tanaka, K. Onoda, H. Ohta, M. Watanabe, Y. Itoh, M. Tsurudone, K. Izutsu, T. Yoshida. (1988) J. Biol. Chem. 263: 4523-4526. 8. Leach, K.L., E. A. Powers, J.C. McGuire, L. Dong, S. Jaken. (1988) J. Biol. Chem. 263: 13223-13230. 9. Wilson, T. (1990) Confocalmicroscopy. pp 325-335. Academic Press, San Diego, 10. Halsey, D.L., P.R. Girard, J. F. Kuo, P.J. Blackshear. (1987) J. Biol. Chem. 262: 2234-2243. 11. Uratsuji, Y., P.E. DiCorleto. (1988) J. Cell Physiol. 136: 431-438. 12. Shoji, M., P.R. Girard, G.J. Mazzei, W.R. Vogler, J.F. Kuo. (1986) Biochem. Biophys. Res. Commun. 135: 1144-1149. 13. Ito, T., T. Tanaka, T. Yoshida, K. Onoda, H. Ghta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, H. Hidaka. (1988) J. Cell Biol. 107: 929-937. 14. Jaken, S., K. Leach, T. Klauck. (1989) J. Cell Biol. 109: 697-704. L. Cheever, H. Khaner, P.C. Simpson. (1990) 15. Mochly-Rosen D., C.J. Henrich, Cell Regulation 1: 693-706. 16. Hocevar, B.A., A.P. Fields. (1991) J. Biol. Chem. 266: 28-33. V.A. Ruff, S. Jaken, S. Kaufmann. (1989) J. Cell 17. Leach, K.L., E.A. Powers, Biol. 109: 685-695. 18. Butler, A.P., G.V. Byus, T.J. Slaga.(1986) J. Biol. Chem. 261: 9421-9425. 19. SaLyoun, N., M. Wolf, J. Besterman, T.S. Hsieh, P. Cuatrecasas. (1986) Proc. Natl. Acad. Sci. USA. 83: 1603-1607.

46