Vol.

174,

No.

January

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

15, 1991

Pages

37-42

HORMONE CONTROLLED PHOSPHORYLATION AND DEGRADATION OF CYPZBl AND CYP2ElIN ISOLATED RATHEPATOCYTES Inger

Johansson,

Department

Erik

of physiological S-104

Received

Eliasson

November

16,

and Magnus Ingel~-S~zY

Chemistry, 01 Stockholm,

Karolinska

Institutet

Sweden

1990

Addition of adrenalin to primary rat hepatocytes caused a 3- and 2-fold increase in [32 PI-incorporation into CYP2El and CYP2B1, respectively. Adrenalin also increased the rate of CYP2El degradation at similar concentrations as needed for phosphorylation of the protein (r=0.93), but did not influence the degradation rate of CYP2Bl. Ethanol (75 mM) completely protected from adrenalin dependent phosphorylation and degradation of CYP2E1, but did not influence CYP2Bl on these parameters. Examination of para-nitrophenol hydroxylase revealed that ethanol stabilized the catalytically active form of CYP2El. Insulin treatment caused a stabilization of CYP2E1, but did not affect CYP2Bl degradation. It is concluded that degradation of CYP2El is the subject of honmmal control, whereas CYP2Bl decomposition is acccmplished in a different and a less regulated manner. 0 1991Academic Press,1°C. The hepatic

microsomal

the environment. inducible other 2),

Thus, many of the various

by their

inducers

own substrates.

can influence

the stability

translation

effect

It

(4) or the stability described

of the ligands

the P450 substrates gene transcription

the rate

that

that

at concentrations

to the enzyme (5).

under

in vitro

or (1,

of protein

specific

ligands

in hepatocyte which parallels

to

CYP2El#

cultures

The CYP2El substrates

conditions

(5,

the binding

enzyme from a glucagon and cAMP-dependent phosphorylation reaction

P450 are

of the enzyme (5-8).

the enzyme from degradation is achieved

of specific

mRNA (3),

to changes in

forms of cytochromes

appears that

the rate

of the specific

We have recently protect

moncoxygenase system is adaptable

causes hem loss.

can

6).

This

affinity

also protect on Ser12',

the a

The

#The nomenclature for cytochrm P450 used is as proposed in Nebert, D.W., Gonzalez, F.J., Coon, M.J., Estabrook, R.W., Feyereisen, R Guengerich, F.P., Gunsalus, I-C., Johnson, E.F., Loper, J.C., N&on, D.R., Sato, R., Waterman, M. and Waxman, D. J. (1991). DNA Cell Biol, in press.

Vol.

174,

No.

phosphorylated the

enzyme is

noncovalently

CYP2B1,

then

modified

In the present stimulation

BIOCHEMICAL

1, 1991

but only

investigation,

opposite

effect

affects

BIOPHYSICAL

apparently

protein

of hepatocytes

AND

degraded

at a higher

that

rate

than

adrenalin

phosphorylation

CYP2El degradation,

regarding

COMMUNICATIONS

(6). we describe

causes

RESEARCH

of both whereas

CYP2El

insulin

and

has an

CYP2El degradation.

EXPERIMENTALPRCXXDURES Materials. Collagenase (type IV, lot 58F-6832), adrenalin (lot 119C-05811) were obtained from Sigma. Porcine sodium Insulin (Lot 050487) was purchased from Lilly. Antisera against CYP2El and CYP2Bl were prepared as previously described (9) and have been characterized before (9, 10). Methods. Hepatocytes were prepared from male Sprague Dawley rats (170 g), previously starved and treated with acetone (5 ml/kg) for two days. This treatment is known to increase the hepatic level of CYP2El about lo-fold to approximately 0.4 nmol/mg and cause induction of CYP2Bl to about 0.7 nmol/mg of microsomal protein (9). Culturing of hepatocytes from starved and acetone treated rats on collagen rapidly causes degradation of CYP2El mFUJA which is declined to about 20% of the original level after 1 day of culture and is unmeasurable after 3 days (5). Examination of cytochrome P450 phosphorylation was carried out as previously described in detail (6). In brief, isolated rat hepatocytes were suspended in phosphate-free buffer, and subsequently transferred to 60 mm culture dishes for 30 min preincubation at 37' with 100 uCi [32P]-phosphate (Amersham)/l.Ei ml and, where indicated, ethanol 75 mM. Cells were then challenged with indicated concentrations of adrenalin or insulin for additional 30 min before termination with ice-cold TES-buffer as described (6). Microsornes were prepared as previously described (5) and immunoprecipitated with anti-CYP2El or anti-CYP2Bl-IgG. The resulting precipitates were washed and subjected to SDS-PAGFI with subsequent into autoradiography of dried gels. Incorporation of [ P]-phosphate cytochrome P450 was analyzed by intensity-scanning of autoradiograms as described elsewhere (6). The same preparations of hepatocytes were also cultured under serum free conditions for two days, essentially as described by Eliasson et al. (5). The levels of cytochrane s CYP2El and CYP2Bl were examined by Western blotting of the microsomal fractions (9, 10). Paz-a-nitrophenol hydroxylase activities were measured on microsanal preparations frcin the hepatocytes, essentially as described by Koop (11). RESULTS AND DISCUSSION Previous

experiments

causes

increased

ser12g

(6)

isolated increased effect

phosphorylation

and Ser128 from

in isolated

starved

(12),

of 100 nM (Fig

respectively.

of both 1).

have shown

that

glucagon

of CYP2El and CYP2Bl at a single

and acetone

phosphorylation

hepatolytes

treated

Stimulation rats

of hepatocytes,

by adrenalin,

CYP2El and CYP2BI at a half

The dose response 38

curves

site,

were

similar,

caused maximal although

Vol.

174,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A CYPZB

CYP2El

-

A

---

A+E

G

----

c

A+E

A ---/I

1

G I

CYPBEl

CYP2Bl

phosphorylation

phosphorylation

C I

I

I

T

1 T

+ Ethanol

CW2El

protein /

0

I

0.05

CYP2Bl I

0.5

I

I

5

10

I/ 0

protein I

I

I

I

0.05

0.5

5

10

pM Adrenalin Fiq.1. Effect of adrenalin and ethanol on phosphorylation and degradation of CYPZEl and CYPZBl in isolated hepatocytes frau rats starvedandtreatedwithacetane. A. Autoradiogram of SDS-gel electrophoretic analysis of CYP2El (left) and CYP2Bl (right) kmmxioprecipitates after stimulation of the hepatocytes with adrenalin (10m5M, A), adrenalin and ethanol (10m5M & 75 mM, respectively, A+E), Glucagon (5x10-'M, G) or treated with vehicle (C). Arrow indicates position of CYP2El and CYPZBl, respectively. B. Phosphorylation and degradation of CYP2El (left) and of 75 CYP2Bl (right) in the presence (O---O) or in the absence (o---o) mM ethanol. The extent of phosphorylation was evaluated after adrenalin treatment for 30 min, whereas degradation was investigated after 48 hours of culture. The amount of CYPZEl and CYP2Bl was determined by Western blot analysis of isolated microscmes and are expressed in relation to the level present in cell cultures treated with vehicle alone. The data shown are mean + SEM of four different experiments. 39

Vol.

174,

No.

the

increase

lB,

the

1, 1991

BIOCHEMICAL

of phosphorylation

degradation

concentrations

rate

that

between

the two

adrenalin

did

caused

(Fig

adrenalin-dependent

whether

the

active,

microscmes

rate

1B).

with

difference CW2El

Thus,

cultured than

in hepatocytes

it

CW2El.

is

evident

These

results

for

CW2El

mechansim Table

I.

CYP2El

with

of CW2El that

ethanol,

does

are compatible breakdown,

In order

with

involving

to investigate

hepatocytes

Table

I,

levels

indicate

of the

(see

functional

previously

an initial

the

(25 mM). This

hepatocytes the

and

30-fold

higher

and might

the

influence

was catalytically

a-fold

stabilize

not

from

to provide

of ethanol

from the

same

I).

was about

in the control

ethanol

but did

from

in the presence

treated

of apo-forms

evident

activity

expected

CYP2El

treated

variously

As is

at the

of CYP2Bl degradation

been shown

(11).

Fig

By contrast,

1, Table

by ethanol,

hydroxylase

was larger

presence

CYP2El

para-nitrophenol.

in hepatccytes

(Fig

from

coefficient

protected

has previously

from

As seen

was 0.93. the rate

of CW2Bl

for

COMMUNICATIONS

by adrenalin

and degradation,

prepared

were

CW2El.

cunpletely

of CYP2El stabilized

of para-nitrophenol

faster

events

Ethanol

reaction

form

RESEARCH

The correlation

influence

hydroxylase

catalytic

incubated

stimulated

or degradation

Para-nitrophenol

with

phosphorylation.

phosphorylation

phosphorylation

a specific

was higher

significantly

in the hepatocytes

BIOPHYSICAL

of CYPZEl was i ncreased

adrenalin

not

AND

below). form of

proposed loss

of heme (6).

Para-nitrophenol hydroxylase activity and relative amount of CYP2Bl. as determined by Western-blotting, in micmscmes isolated frun hepatccytes cultured for 48 hours

and

Hydroxylase nmol/mg,

Treatment None Adrenalin, 5 PM Ethanol, 25 n-&l Adrenalin, 5 PM + ethanol, 25 nN Hepatccytes

were

isolated

activity min

CYP2El (%)

CYP2Bl (%I

0.03+0.02 n-d. 0.96tO.07

100 68+8 8325156

100 9625 10024

1.15eo.35

844+157

99+2

frun

rats

starved

and treated

with

acetone

as described under Methods. The results represent meanvalues + SEZM of three different experiments performed protein is related to the concentration vehicle only. n.d., not determined.

40

in triplicate. in cultures

The amount of CYP treated with

Vol.

174,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

yMInsulin Fiq.2. Effect CYP2El (o---o)

of insulin on the rate of degradation and CYP2Bl (O---O) in isolated rat

of cytochrcme hepatocytes.

The cells were incubated with indicated amounts of insulin for 48 hours and the amount of CXP2El and CYP2Bl was subsequently determined by Western-blot analysis of isolated microsomes. The data shown are mean +SEMof three different experiments. Stimulation

of the hepatocyte

stabilization

(Fig

2).

The half

of the present

whereas CYP2Bl is not affected

separate

control

mechanisms for their

findings

in vivo.

of 7 h and 37 h, respectively from the fast

degraded only differences catalytic acetone, half

life

activity

show that

required of CYP2El

the rate

(7).

degradation.

(7).

with

This is in agreement

and protein

By contrast,

a half

concerns the fact decline

that

of the enzyme (14).

life

half

lives

induced CYP2Bl is Further

CYP2Bl dependent after

down regulate

CYP2El protein,

indicating

Thus, taken together 41

to protect

of 37 h (13).

concomitant

whereas CYP2El dependent activities than the corresponding

and

by these hormones. This indicates

Acetone has the capability

t urnover

of

by adrenalin

CIP2El is degraded with

at a slower rate

in vivo

of apo-pools

of insulin

influenced

insulin,

the protein

of CYP2Bl

change in the extent

investigation

of CIP2El is significantly

previous

the rate

caused

could he seen (data not shown).

The results degradation

insulin,

maximal concentration

high (250 nM) and no significant

phosphorylation

with

with

of CYP2E1, but did not affect

degradation was quite

cultures

induction with

with

a shorter

the formation

these data indicate

Vol.

174,

No.

BIOCHEMICAL

1, 1991

the existence

of at least

cytochrcme

P450 degradation,

mechanism

and one substrate

protein

is degraded

AND

BIOPHYSICAL

two principally

and hormone

and substrate insensitive

(14,

COMMUNICATIONS

mechanisms

different

one hormone

in the lysosanes

RESEARCH

for

regulated

fast

mechanismwherethe

15).

ACKNOWLEDGMENTS We areindebtedtoMrs Asa Johansson for excellent technical assistance. This work was supported by grants from Lars Hierttas Svenska Lakarsallskapet and from the minne, Magn Bergvalls Stiftelse, Swedish Medical Research Council. REFERENCES 1. Rangarajan, Sci

P. N. and Padmanaban,

(USA)

2. Sogawa, K., Y.

(1986)

G. (1989)

Proc.

N&l.

Acad.

86, 3963-3967.

Fujisawa-Sahara, Proc.

Natl.

3. Song, R.L.,

Yamane, M., and Fujii-Kuriyama, USA. 83, 8044-8048. Hardwick, J.P., Park, S.S., Veech, H.V. and Gonzalez, F.J. (1987) Mol.

Acad.

A.,

Sci.

B-J., Matsunga, T., Yang, C.S., Gelboin Endocrino~. 1, 542-547. 4. Kim, S.G., States, J.C. and Novak, Metabolizing

Enzymes:

Genetics,

R.F.

(1990)

Regulation

in Drug and l"oxicology

(Ingelman-Sundberg, M., Gustafsson, J.-A. and Orrenius, S, Eds.) p. 73. 5. Eliasson, E., Johansson I. and Ingelman-Sundberg, M. (1988) Biochem. Biophys. Res. Cm. 150, 436-443. 6. Eliasson, E., Johansson, I. and Ingelman-Sundberg, M. (1990) ProC. Natl. Acad. Sci. (USA) 87, 3225-3229. 7. Song, B-J., Veech, R.L., Park, S-S., Gelboin, H.V. and Gonzalez, F.J. (1989) J. Biol. Chem. 264, 3568-3572. 8. Watkins, P.B., Wrighton, S.A., Schuetz, E.G., Maurel, P. and Guzelian, P.S. (1986) J. Biol. Chern., 261., 6264-6271. 9. Johansson, I., Ekstrtim, G., Scholte, B., Puzycki, D., J&nvall H. and Ingelman-Sundberg, M. (1988) Biochemistry 27, 1925-1934. 10. Johansson, I. and Ingelman-Sundberg, M. (1988) Cancer Res. 48, 5387-5390. 11. Koop, D. (1986) Mol. Phamacol., 29, 399-404. 12. Pyerin, W. and Taniguchi, H. (1989) EM730 J 8, 3003-3010. 13. Shiraki, H. and Guengerich, F.P. (1984) Arch. Biochem. Biophys.

235,

86-95.

14. Ronis, M.J.J. and Ingelman-Sundbrg, 19, 1161-1167. 15. Masaki, R., Yamamoto, A. and Tashiro, 104, 1207-1215.

42

M. (1989) Y. (1987)

Xenobiotica

J. Cell.

Biol.