Elution of the regulatory subunit of cAMP-dependent protein kinase Type I isozyme derived from epididymal fat within the Type II isozyme chromatographic peak

Vol. 112, No. 1, 1983 April

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

15, 1983

Pages

214-220

ELUTION OF THE REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE TYPE I ISOZYME DERIVED FROM EPIDIDYMAL WITHIN THE TYPE II Alvin

School

ISOZYME CHROMATOGRAPHIC PEAK

M. Malkinson,

Jeanne

M. Wehner

of Pharmacy,

University

*Dight

Institute

Genetics

for

and Cell

Deborah

February

S. Beer,

and J. R. Sheppard* of Colorado,

Human Genetics Biology,

Boulder,

CO 80309

and Department

University

Minneapolis, Received

FAT

of

of Minnesota

MN 55455

28, 1983

No CAMP-dependent protein kinase activity is found upon DEAE-cellulose chromatography of mouse fat extracts at the low salt concentration characterThe RI detected in fat extracts by photoincoristic of the Type I isozyme. poration of the analog, 8-N3[ 32P]cAMP, elutes within the high salt Type II The multiple charge variants of this photolabeled RI which isozyme peak. can be resolved by two-dimensional gel electrophoresis are similar to those of the histoptypically-related cultured cells, SV3T3 and 3T6, which do contain Type I kinase isozyme activity peaks. This high salt-eluting RI may be part of a Type I holoenzyme whose elution properties are altered by interactions with other substances present in the extract.

CAMP-dependent es,

called

Types

lulose

columns

units

and vary

on what

these

These

roles

(4,5),

kinase

salt

(Type

0006-291X/83

II)

.

enzymes on the

their

have

during

usually peaks

traverse

based

(6),

method

$1.50

Copyright 0 I983 by Academic Press, Inc. AN rights of reproduction in any form reserved.

214

(2).

from DEAE-cel-

the

relative

re-

speculation

Such changes

at low

the

in the proporinclude

tranformation

the sizes

DEAE columns

(C) sub-

Although

on alterations (3).

class-

catalytic

and neoplastic

measures

isozymic

been delineated,

by comparing

from

major

elution

subunits

changes

estimated

An alternative

of their

have not

largely

two

or identical

(R)

isozymes

eluting

into

similar

physiological

cycle

fall

order

regulatory

may be is

cell are

activity

based

of these

isozymes

proportions

tein

in

functions

of the

kinase

The isozymes

only

--in vivo

development

I and II,

(1).

spective

tions

protein

of the (Type

I)

amounts

(7). two proand high of

the

Vol. 112, No. 1, 1983

photoaffinity

analog Although

unit.

can RI

TyJje

II

st:lte

elute

zyrle act-ivity

peaks

a variety

(11-13). peak

would

solely

on

the

concentrations.

contain

an that

RI--subunits

--Materials

RI

no

which

apparent contained

and

DRAB

from

the

salt

of

certainly

cloud

proportion

of Herein

elutes

an

that

within

I

isozyme

the

mouse Type exist

peaks

from

by

I

isozyme

Type

I

and

dissociation

within

peaks

the

differences

Type the

to

of

activity

show

the

addition

isozyme

R-sub-

detectable

between

interpretation

these

each

uncharacterized in

I

is

of

elutes

of

the

solely

Type

outside

C-subunit

Type

we

structural in

columns

into

which

RI,

concentrations

presence

RESEARCH COMMUNICATIONS

incorporated

(9),

RI-subunits

of The

(8),

agree

from

while

(lo),

at

often

elute free

AND BIOPHYSICAL

8-N3[32P]cAMP

assays

also

isozyme

and

CAMP,

dissociated

peak

re1.y

of both

photolaheling peak.

BIOCHEMICAL

the

those to

Type

Type

I iso-

II

kinase

studies

which

estimate

relative

epididymal

fat

II

kinase

peak,

Rl

those

between related

isozyme this

extracts

and

sources.

Methods:

Epididymal fat was removed from adult BALB/cByJ mice and Chromatography. 7 honogenlzed in 10 mM potassium phosphate, pH 7.0, containing 1 mM EDTA (2 vol/ pm wet wt.). The crude homogenate was passed through silk to remove excess lipid, and centrifuged at 27,000g for 35 min to obtain supernatant fractions. Chromatography on DE-52 cellulose (Whatman) equilibrated with this same buffer was done according to the method of Corbin et al. (l), as previously modified (14). Prior to chromatography, the extract was preincubated with ATP and MgC12 to final concentrations of 0.15 mM and 3.5 mM, respectively, to facilitate association of the Type I holoenz,yme. After 30 min at 30°C the extract was chromatographed on a Sephadex G-25 column (30 X 0.9 cm) and fluted at 4°C at a flow rate of 30 ml/hr. Proteins eluting in with homogenization buffer the void volume, as determined by absorption at 280 nm, were pooled and 20-30 mg of protein was applied to the DEAR-cellulose column. Assays. Protein concentrations were determined according to Lowry et -_ (15), with crystalline bovine serum albumin used as a standard. Protein --al. kinase activity was assayed with a 250 ~1 reaction mixture containing 6.7 mM 2.0 mM 2-(N-Morpholino) ethane-sulphonate (MES) buffer (pH 6.5), 233 I.cg MN2, Type II A mixed histone, 0.23 mM (Y-~'P]ATP (40 Ci/mmole), and a 25 pl aliquot with or without 3.3 PM CAMP. After 30 min at from a DEAE-cellulose fraction, 100 ~1 aliquots were transferred onto filter paper and washed with tri3O"C, The photoincorporation procedure used chloroacetic acid as described (16). previously (17) was slightly modified. Twenty ~1 of a column fraction and 1 U1 of Ml'S buffer were added to a 5 ~1 aliquot of 8-N3['2P]cAMP to obtain a final analog concentration of 125 nM. These components were mixed with an The samples were incubated at 35'C for 30 min in airstream on a spot plate. the ligand and any endogenous CAMP altJe dark to promote exchange hetween ready hound to regulatory suhunits and were further incubated for 30 min at Photolysis with IV (254 nM) was done at 4°C with a DVS-54 4'C in the dark. Mineralight handlamp held at 12 cm for 15 min. The reaction was terminated with 26 ~1 of an SDS-containing stop solution (18), and the photolabeled Protcins separated on P-12"/ linear gradient denaturing gels.

215

Vol. 112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Gel Electrophoresis. Two-d$?ensional electrophoretic separation of crude was done as follows. Epididymal fat extracts photolaheled with S-N3 [ P]cAMP was homogenized in 0.32 Y sucrose and post-mitochondrial fractions prepared as ahove. Roller bottles of PALR 3T3, SV3T3, and 3T6 cells were grown in Dulbecco’s modified Eagle’s medium, supplemented with serum. The cells were rinsed twice with cold 0.32 M sucrose, scraped and centrifuged at 900g. The pelleted cells were then sonicated for 10 set at 1.5 watts on a Bronson sonicator, and frozen for later assay. Twenty ~1 of extract containing 20 ug of protein was photolabeled using an S-N3 [ 32PjcAMP concentration of 750 nM and the reaction terminated with 26 u1 of lysis buffer (19). The O’Farrell (19) electrophoretic procedure was modified as described previously (17). First-dimension gels were run for 6400 volt-hrs. Samples were overlayed with 5% Nonidet P-40, 8 M urea, and 1% ampholines, composed of 0.8% of the pH 5 to 7 ampholine and 0.2% of the pH 3.5 to 10 ampholine, both from LKB Instruments, Inc. Second-dimension gels were 8 to 12% linear gradients. Dried gels were placed on DuPont Xray film with DuPont Quanta-111 intensifying screens for l-2 days at -75’C for autoradiographic detection. Results

and Fig.

tein

Discussion: 1A illustrates

that

activity

measurements

kinase

activity

elutes

enzyme

at When

(1).

photolaheled, in

on

its

suggest he

size

associated

hound

CAMP

elutes

from

Type

II

the

void

Type

II

and

activity volume peak

This at

the

RALB

is

the 3T3

pI

range

(5.4

the

RI

and

RII

subunits

more

salt

peaks

(10);

3.

(14),

was

observed;

4.

(M.S.

Butley

has

cells

not

salt-eluting

characteristic

peak

yet

been

RI

was I

a Type

C-subunit In

and

A.M. in

other

isozyme

I

with

position.

are

photolabeled RI

is

Type

II

allows

charge

variants.

isozyme

kinase

bas-

peak

may

extract

was

preincuhated

release

of

endogenously

Free to

RI

typically

the

Type

which

the

RI

Malkinson,

I

and

elutes

eluting

at

in

the

unpublished

re-

systems. RI-subunits

which

elute

Two-dimensional

proteins

216

holo-

considerations

this

2.

lung

II

extract

as

activity,

mouse

compared

R-subunit lack

of

fat the

intermediate

tested

photolabeled

photolabeled (7,21,22)

No

the

(14,20);

concentrations

Type of

facilitate

reassociation

at

a holoenzyme

The

prothis

Type

following in

1.

the

protein

The

5.6).

by

extracts;

of

this

present

to

subunit

separation various

-

holoenzymes:

chromatography

columns

this

high

trophoretic of

and

encourage

DFAF

although

suits),

K)

for from

of

detected

fat

one-third

Identification

RI.

he

of

typical

than

is

to

can

derived

greater

as

peak

range fractions

lB),

C-suhunits

prior

DEAF: chromatography

peak

with

E!pATP

single

upon

column

(Fig.

(49 hoth

a

concentration

individual

this

that

with

salt

however

material ed

the

only

qualitative Both activity

eleccomparisons

fat peak,

tissue but

and con-

Vol. 112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FRACTION NUMBER (A) Protein kinase activity in the 1 presence (U) or absence --Figure (C-@) of CAMP, and (B) 8-N3[32P]cAMP incorporation into RI (@----O) and RI1 ( l -. ) in DEAE-cellulose fractions collected after chromatographic elution of extracts from epididymal fat. The NaCl concentrations were estimated by conductivity measurements of each fraction and extrapolation from a standard curve.

ta:n

RI-subunits

This

is

detectable

interesting

prajducts

that The

ha*;

anchorage

tain

an

SV3T3

autoradiogram

trophoretic ovarian

viral dependence

from of

patterns

are (24),

I

and

kinase

one

3T6,

similar

3T6, to

lung

the

(17),

other, and

217

potential

BALB

can

3T3

gel

of

and

epididymal

mammalian

and

22).

rise

each

proteins fat.

those

described

brain

(25).

con-

2 is

Fig.

(22).

to which

SV3T3,

photolabeled

to

give

transformant

peak

and

1B;

differentiated

cells

activity

SV3T3,

each

(Fig.

a spontaneous

between isozyme

3T3,

of

precursor

a two-dimensional

BALB

mouse

photolabeling

mesenchymal

intermediate

Type

extracts

follicles

are

transformant

prepared

from

8-N3[32P]cAMP

adipocytes

multipotent

demonstrable

a

tained

since these

(23).

by

ob-

These

elecfor

When

an

rat large

Vol. 112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

54K 4 49K4 45K 4

MW 54K+ 4QK+ 45K 4

Figure 2 Autoradiogram of proteins from (A) BALR 3T3, (B) 3T6, (C) SV3T3 and (D) epididymal fat extracts which were labeled with 750 &I 8-N3[32P]cAMP and fractionated by two-dimensional gel electrophoresis.

amounts

of

iants

g-N3[ 32P]cAMP

ranging

central

in

spots

of these

migrates

RI1

presence

of

of

present

the in

does

42-44K

charge

lines

is

ment

derived

brain

(25)

at

not

of

since the are

prominent in

the

RI,

observed

label. are

the

but

most

These

of

in

labeling

labeled

in

the

SV3T3

8-iV3[32 PIcAMP-labeled

also

a single

labeling

of

less

var-

the RI,

two only

fat

218

are the of

these

but The main in

frag-

endogenously of protease

proteins.

from

extracts,

extract.

probably

the

Labeled

proteolytic

presence

extracts

R-subunits

37K spot.

suggesting charge.

represent

fragments

in

from

with

p1 dimension,

likely

found

labeled

charge

samples,

non-identical

homogenization amount

discrete

in all

With

along

42-44K

four

detected.

similar

(26,27).

variants most

most

elongated

37K and at

reduce

into

are

species

Proteins

samples

detected

is

the

R-subunits

and are

fragment

to 5.6

charge

RII

these

hibitors

5.4

as a spot

two

variants

ments

incorporated

incorporating

two or three

charge

pI from

are

the only

Multiple cultured a single

proteolytic

mouse

in-

lung

cell 37K frag-

(17)

and

BIOCHEMICAL

Vol. 112, No. 1, 1983

The

affinities

similar

for

of

the

affinities

fat

nor

and

the

cell

density

was

fron

0.4-2.5

mg/ml

the

charge,

and

thereby

ty

of

the

as

resolved

charge by

peak

may

by

MgPTP inp

in

(29))

and

capacity

of

birding This

of

is

cA?fP-dependent su’>unit

structure

stlldies,

along

R-,:uhuni

peaks Type

I

of

8-N3

11s ,

functional

and

even

as

subcellular

status

R-subunits

be

valid

On

the

into in

the

concentration

the

suggested

by

high

salt

lack

characterizing

titration accurately

since

similari-

the

the

relative

sizes

of

type

hand, R-subunit

II

the

activ-

the

relative

can

be

modi-

concentrations can

affect

of

stoichiometry

has

been

and and

quantitative

with

antisera the

net

concentrations

the

such

describe

the

of on

which

R-subunits

the

explanation

endogenous

A

sub-

This

other

of

analog.

within

possibly

columns.

each

all

chromatography

to

when

and

solely

distribution

immunochemical

of

not

(30))

for

kinases

required

the

conformation

and

based

and

purified

useful

be

protein

Estimates

[32P]cAMP

to

most

may

is

low

isozyme.

perhaps

with

RI

the

may

the

DEAE

affected

its

DEAE

of

(28)

for

(30).

from

adenosine

R-subunits

also

Neither

substances,

influence

isozymes

strength

[32 P]cAMP

8-N3

analog

MgCTP

were

cellular

at

kinase

ionic

are

shown).

the

electrophoresis.

the

[32P]cAMP

not

varying

may

eluting

incorporation changes

other

position

activity

contain

8-N3

protein

by

properties

protein

kinase

quantitative fied

elution

variants

the

(data

RESEARCH COMMUNICATIONS

assay.

enzyme,

two-dimensional

of

samples

or

and

kinase its

binding

photolabeling RI

protein

CAMP-dependent ity

the

for

bound/mg

growth

ion-exchange

RI

prcportions

ligand

during

in

atypical

cell

of

hetween

for

the

cultured

varied

strates

subunits

RI

amounts

Interactions

for

the

AND BIOPHYSICAL

of the

bindin

the

described properties

(31). of

modifications

the

of

R-

photolabeling prepared

against

concentrations

and

cell.

--Acknowledgements: This sota

We thank work was Leukemia

Martin S. Butley for his supported by USPHS grant Task Force (to J.R.S.).

valuable ES02370

(to

comments A.M.M.)

on

this manuscript. and by the Minne-

References: --1.

Corbin, 225.

J-D.,

Keely,

S.C.

and

Park,

219

C.R.

(1975)

J.

Biol.

Chem.

250:218-

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B.E. (1977) Meth. Enzymol. 46:339-346. Walter, IJ. and Greengard, P. (1978) in Handbook of Experimental PharmaVol. 58, Eds. Nathanson, J.A. and Kebabian, J.W. COl%Y, Springer-Verlag, New York, pp 479-505. Walter, U., Costa, M.R.C, Breakfield, X.0. and Greengard, P. (1979) Proc. Natl. Acad. Sci. USA 75:3251-3255. Walter, U., Uno, I., Liu, A.Y.-C. and Greengard, P. (1977) J. Biol. Chem. 252:6494-6500. D.L. and Strittholt, J.T. (1981) Biochim. Biophys. Acta. 675: Friedman, 334-343.

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220