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The protein kinases of rat liver nuclei
Vol.
93, No.
April
BIOCHEMICAL
4, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
29, 1980
Marianna Sikorska, F’. Roy Walker and
1196-1203
Animal and Cell l~hysiology Group Division of Biological Sciences Nat ional Research Count i 1 of Canada Ottawa, Canada Kl:l OR6 Received
March
11,
1980
Soluble proteins of rat liver nuclei I\‘ere applied to a DIALScphacel column and eluted \iith a linear gradient of 70-600 mill NaCl. Three major classes of nuclear protein kinase were identified. There Ivcre types I and II c:UIP-dependent protein Ainasc, a Ca-cnlmodul in-dependent protein hinasc, and a third enzyme which 1~31; stimulated by c.\biI’-dcpendcnt protein hinase inhih i tor. ‘The
patterns
protein
of
PK
(protein
phosphor~lation
division
cycle
suggesting
change
[l-3).
th;1t
hinnsc)
There
1'6s
a h-C)
s.
and
i 1’ 3 t e cl n 0 n - n e 0 I) 1 3 s t i c cells
cl\,iIP
surge
,\lorcovcr,
in
deprivation
almost
immedintel!~
fcx
as
cation
requirements
nuclei
(b-lo),
IlLlclcus
ha5
dependent
Cl
cultivated
calcium
,I5
late
example,
can
tlbo
to
need
induced
the
initiation
in
regenerating calcium
in
late
C!l,\lP)
and
a.5 tkelvc
or
t).pe
forms
growth-
evidence of
I)?;.1 liver
and
a brie1
(A’,. Gl
initiate
I,!, calcium,
as man)’
of
DKA synthesis
to
nuclear their
lines
both
arrested
phase
by
DNA synthesis, IT
c.lblP-dependent
of
PK liith
PI<
di f‘fcren
:i n d 5 u h s t r :I t c p r c f c r c II c c 5 h :I v e h I i’ II f 0 II n d i II but
always
the
presence
been
1’6 activity
of
f : 1980 fly .A cadem,c Prec.5, of reproduction in an-v f‘orm
ciZbiP-cleI)clldcIIt
controversial has
Inc. resertped.
FE.5 in
(3,7,9,11).
recently
been
0006-291X/80/081196-08$01.00/0 Cop-vngh All righrs
in
initiate
cells be
role
and
through
several
hcpatocytes
phase liver
pass
also
synthcsi c 111 t
1’11u 5 , for
as cells arc
play
may
activity
1196
found
flolicve in
thymic
the r , cANI’lymphocyte
Vol.
93, No.
nuclei several
4, 1980
1112). types
cAlilP-dependent by
BIOCHEMICAL
In
this
0P Pli Pk:,
Ca-calmodul
AND
BIOPHYSICAL
communication in and
rat
liver
we
report
nuclei,
a hitherto
RESEARCH
the
presence
including
unnoticed
COMMUNICATIONS
trvo
PK which
is
of types
of
stimulablc
in. IlATL:.RIALS
Bovine serum albumin, CAMP, cGblP, calf thymus histones Sigma type 11,2 (mixed) and Sigma type V-S (111) , beef heart protci n kinasc inhibitor (PKInh), salmon sperm protaminc, 3-isobutyl-1 methyl-xanthinc (IBNX) were obtained from Sigma Chemical Co. (St. Loui s , MO, USA) ; DEAL-Seyhacel ~3s obtained from l’harmac ia (Uppsala, Sweden) ; [ 2,8fl]cAMP (.37 mCi/mmole) was obtained from Amershan Corp. (Oahvillc, Ontario, Canada) . [ y- 32 P].A?‘P was prepared according to Flynn and Chappell (13), and calmodulin was purified to homogeneity from rat testis accordj.ng to ~Iacblanus (14)
Isolation -__~-extract.
of
nuclei
and
preparation
of
soluble
nuclear -___
p rotein -
Liver tissue from specific pathogen-free male Sprague-Uawley rats (1’30-110 g) c~as homogenized in b volumes of buffer A (50 mbl J’ris-HCl, 25 mM KCl, 5 mbl blgC12, 250 mi4 sucrose, pfl 7.5) and then centrifuged at 700 gav for 10 min at 7°C. The pellet was washed with buffer A, and resuspended in 5 volumes of buffer A containing 1: Triton ly-1OU. After centrifugation the pcllct icas \\,ashed again with buffer A. The nuclear preparations were slightly contaminated by mitochcndria (about 5”, of total mitochondrinl mononminc osidasc and s:l:-i:ini; dehydrogenasc activity was associated with nuclei), but contained neither lysosomcs (asscsscd hy Eluorescencc microscopy after staining with acridinc orange ) nor mi crosomt‘s (indicated by no NM~Pll cytochromc c rcductasc activity). h’iuclci hiore extracted with (1.h I\1 NaCl in buffer 13 (20 m,\l ‘Pris-ilC:l, 3 mN &Cl z 3 mbl l:l)TA, 50 mb1 ~-mcrcaptoethn~iol , pll 7. 5 j . The nuclear suspension \kas centrifuged for 15 min at 20,000 gav, the pellet lizis discarded, and the supcrnatant diluted lo-fold with buffer H. The precipitate formed in the diluted supcr;latant 1~:1s removed by celltrifugnt ion at 10,000 ~av for 10 min, and the clear supernatant containing SO”, of the original nuclear proteins and about 80’: of the total nuclear PK activity was applied to a III’AtiSephncc\l column, as described in the figure legends. PK
(Protein hinase) assay Pli activity was assa)~cd according to Tahai et al. (1 sj . ?‘hc assay mixture (0.15 ml) contained: 7.5 nmole [y-‘*P]ATP (2 x 10” cpm/nmole), 7.5 pmole Z-mercaptocthanol, 1.25 or IS.75 Umolc 100 pg of mixed histonc, historic 1lp.C 1 2 ) 5 umolc Tris-klC1 (pll 7.51, and the appropriate enzyme fraction. The 111 or ;~rotai.line, mixture was incubated for 5 min at 3O”C, and the react ion was Bovine serum stopped by adding ZO”,, trichloroacetic acid (‘l‘(X). albumin (0. 5 nig) has added a.5 a carrier. The acid-prccipitnblc in 1.0 N Ya1)fl arid matcrinl was dashed three times h) dissolving rc-prccipitnting in TCA. Ihc radioactivity of 321’ incorporated
1197
Vol.
93, No.
into liquid
the
Other
BIOCHEMICAL
4, 1980
protein scintillation
substrates counter.
AND
was
BIOPHYSICAL
measured
RESEARCH
with
COMMUNICATIONS
a Beckman
LS
255
procedures
cAblP-Scpharose was prepared as described by Dills et al. (lb). The coupled density of immobilized N.;“- I12N(CH2) 2-cAb!P was 6 ~molc/ml of scttlcci gel. The regulatory subunit of cAbif’-dependent PK attached to this gel was isolated and purified by elution with 1 ml\1 cGi\lf’, concentration on an Amicon filter (Xb15) and extensive dialysi 5. Protein content was measured by a dye-binding assay (Hio-Rad: 17). RESULTS The classical from
cytoplasmic forms
DEAE-Sephacel
AND DISCUSSIOh’
extracts (I
and by
of
II)
of
100
and
liver
cells
cAMP-dependent 200
mbl NaCl
contained
the
PK,
were
\ihich
respectively
l
x
two eluted
(18).
With PK M,. Withad PK Inh.
,
Figure
1.
The protein kinases from the cytoplasm and nuclei of liver cells. 40 mg of cytoplasmic (A) or nuclear (B) proteins were applied to DEAE-Sephacel columns (0.9 x 30 cm] previously equilibrated with buffer B. The column was washed with the same buffer, and elution was carried out with 100 ml of a linear gradient of NaCl (0. - 600 were collected. mM) , and 2 ml fractions Enzymatic activity was measured with 5 mM Mg'+, mixed histones, 1O-6M CAMP, 2 mMIBMX, and with or without 10 pg of protein kinase inhibitor (PKInh). 1198
The\, ,
Vol.
93, No.
4, 1980
BIOCHEMICAL
phosphorylated
mixed
~crc
inhibited
by
(Fig.
14) -
On the
of
I’K activity
the
same
the
latter,
nuclear PKInh
in
other
hand,
nuclear
was
the
there
extracts, as
inhibited
activity
BIOPHYSICAL
RESEARCH
presence
of
cA)lP-dependent
concentrations were
(Fig.
histones
a specific
in
NaCl
AND
by
eluted
by
5 mbl big*+,
PK inhibitor,
uere
three
two the
COMMUNICATIONS
of
PKInh
distinct
which
peaks
were
eluted
cytoplasmic
enzymes
PKInh
(Fig.
The
300
mFI NaCl
1B). and
was
and
by
and,
third
like
type
stimulated
of by
1B).
. 7orTMNocl*
-70-600
I
mM N&---t 0 x A
* 70 mM NoCl B
Wim PKkh. WitId PK Inh. [3HmhiP-m
. 0
t
x
WhPKhh.
wacd+md cdfT&A Noaddtion
-&....,.I.......,
0
Figure
2.
IO
20 30 40 FRXW+JMJWEFl
50
60
70
DEAE-Sephacel chromatography of soluble liver nuclear protein kinases treated with CAMP. Nuclear proteins (12 mg) were applied to a DEAE-Sephacel column (0.9 x 30 cm) equilibrated with buffer B. After the column was washed, the first elution was carried out with 50 ml of 70 mM NaCl and lo-'M CAMP, followed by the second elution with 100 ml of a linear concentration gradient of NaCl (70 - 600 mM), and 2 ml fractions were collected. To establish CAMP-binding activity, nuclear proteins were loaded on to the column in the presence of 1 nmole of [3H] CAMP (37 mCi/mmole). Before the first elution, the column was washed extensively with buffer PK activity was assayed with 5 mM Mg' , mixed B. 2 mM IBMX, 10m6M CAMP, with or without histone substrate, 10 ug of PKInh (panel A), or (panel B) with 75 mM Mg'+, protamine substrate, with or without 10 ug of PKInh. In panel B, the fractions were also assayed in the presence of 3 x 10e6M calmodulin and 5 x 10W5M Ca2+ (panel B). 1199
Vol.
93, No.
4, 1980
If
8lOCHEMlCAL
the
two
subunits
(181. of
PIis,
Icould
two
them
the
appropriate
altered
by
the
high
On the
other
described
in
inhibitable and
was
the
phosphorylnte two
types
legend
due
of
Fig.
to
of
3)
was
in
m>l ,\lgs+
protein
requirements the three major
to
they
were
extract
them
(1:).
extract
1). on
3:\)
preferred AS
the
PKInh-
of
(Fig.
\k,hich
(Table
(as
peak
70 mM NnCl ts
subunits
1~~ addition
;I ne\i
by
remainelI
cAMP,
hecause
nuclear
C suhuni
5.0
t\io
[R)
(C)
stimulated
generated
eluted
liberated
CAMP-binding
lilagnesium
not
the
to hy
catalytic
used of
due
regulatory
pi-ohably
treatment
COMMUNICATIONS
stimulahle
distinct
active were
which
histones of
two
concentration
cAbIF
were
been
fractions,
NaCl
activity,
probnhly
into
activities
to
hand,
have
identical
their
RESEARCH
activities
should
nearly
However,
cAl\lP
they
dissociate
and
BIOPHYSICAL
PKInh-inhibitable
cAlzlP-dependent which
AND
to ted,
CXPCC
columns
which
TABLE 1 and substrate preferences nuclear classes of Pi;
Protein
Kinasc
activity
[\)
Substrate 5 Fraction His
tone, (type Histone, (type Protamine
mixed IIA, Sigma) Hl V-S, Sigma)
His
tone, (type Histone, (type Protamine
mixed IIA, Sigma) Hl V-S, Sigma)
Histone, (type flistone,
mixed 11.4, Sigma) Hl
(C
subunit
of
(mFl) 75 c.A;ilP-dependent
75
31
31
60 11
51 10
20 8
13 ?
36
(Ca-calmodulin
stimulable
92
-- .7
70
61 108
41 so
26 1’4
25 175
50
(PKInh-stimulablc
illi)
100
81
56
55
108 50
107 64
38 0 I)
‘7 _I 65
expressed as the percentage of that (in 5 m&l magnes iumj as a substrate.
1200
PI<)
PI0
100
I-raction
is
10
concentration 50
10 II
Fraction
PK activity mixed histoncs
hlagnesium 20
assayed
\,ith
Vol.
93. No.
BIOCHEMICAL
4, 1980
AND
BIOPHYSICAL
RESEARCH
TABLE 2 and regulatory (R) activity of catalytic
Effects of cAblP phosphorylating
COMMUNICATIONS
on the subunit.
subunit
(C)
Pbosphorylating activity i-pm 32P from [~-32P]~7E’/100 misci. hi~tones/S nlin
~fi
C subunit* 16),360 R subunit** 1 , ‘00 !I ) 960 (C + R) subunits (C + R) subunits + cAblE’ 15,150 i Extensively dialyzed fraction 10 (Fig. :A) was the source of C subunit ** ITractions ih-46 (Fig. ?A) were poolecl and K subunits were purified on a cA>EP-Sephurose affinity column were
eluted
ivith
purified
(on
activity
of
This
rat
it
the
forms
\\a~
cells
the
FKs
, these
subunits
of
and
hl cNIF.
10-s
fractio.ls
elutcd
The
fir,st
group
36,
and
~3s
second by
FKInh
of
activity
assayed
and
in
activities (‘Table
Two from
I’lis
eluteci
prohahly,
higher Fig.
the
(Fig.
c.UlF
of
2.61) . (‘l’ahlc
2)
non-proIifcr:lting
of
the
were
same
two
gradient
by
higher
hy fraction one
1B.
The
with
protamine
responsible
activity
fractions
was
Although 1201
fractions
the
1,1),
\\hilc
strongly for
and
in
concentrations. fraction the
stimulated the
thirJ
peah
1~3s similar
a suhstratc,
3?-30
C
70 m>l NaCl
in
profile as
putative
peaked
(I:ig.
46,
the
detected KaCl
those
concentrations.
with
mSI NnCl, FKInh
masked
\Vhen
eluted were
b!~ 100-150
h’nC1
revenleJ
enzyme
the
ZH).
of
of
in
in
FKs
range
were
the
was
1,
same
unaffected
75 mbl big?+
nuclei
cM]P-dependent
the
groups
peaked
were
the
c:lblP-dependent
i:igurc
10
r-c-adding
proportions
activities
which
in
by
different
by
seemingly
one,
turn,
further
recluccd
fraction
and
of
eluted
\vas
lihen
Fli.
minor
the
from
in
3.21 anti,
, substantially
cytoplasm
activities
minor
(! i g.
C subunits
contain
llowever
46-57
column)
0 f cAbIT’-dependent
the
m&l i\‘CrCl
overcome,
that
Th c high of
:70
putative
seems
liwr
and
cWP-Scpharosc
reduction
Thus ,
120
but
r~.hen the
lower
in
fractions
46-58
liere
still
Vol.
93, No.
4, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
TABLE 3 nuclear PK(s) from fraction ZI\) by calcium and cnlmodulin
rlctivation (Fig.
of
pmoles
Basal activity + Ca2 + (5 x 10-51\1) + Calmodulin (10-6iV) + ca2 + + calmodulin + + Ca2 + calmodulin !:CTA (5 x 10-4bl)
the
fraction
(Fig.
50 Since
PK
(19-20)
(211,
also
have
the
been
stimulable \ihen
assayed
(eluted with
not
was
80-120
protamine
as
stimulated
by
calmodulin
together 7-
abolished
to
this
are
contain stimulated
strikingly
1787
PKInh of
protamine
as
PKInh-stimulable
substrate
activity
fraction
46
preparation
(Fig.
an
stimulated
by
However,
PKInh
in
been
found
this
unaffected
histones
in
presence
in
study
maximally
50 was the
a cGPIP-
has
fraction
in
:A).
enzyme
the
in
was
stimulated
such
unnoticed
rnbl NaCl), substrate
might
PKInhby
of
which
not
the
nuclei
10m6bI
5,
20,
cGI\IP or
fractions
active
3))
in
and
5 x 10d5
was
75 mb’
calcium
31-40 75 mizl Fig
and
the
10T6
enzyme’s
chelator
EG’IA
3).
from
proliferntively of
Ca-calmodulin
PKs, or
requirements 1202
inactive
the
activities
PKInh, and
and
liver of
which
substrate
2+
strongly
N calcium
increased
specific
classes
magnesium
in
more
1 and
separately)
(Table
main
was
Thus,
while
CAMP,
PK species
(Tables
(but
that
different
with
than
stimulation
by
799
shown).
X-fold
three
3’17
377
Ca-calmodulin.
We conclude cells
834
since
a hitherto
with
activity
in mixed
(data
There
450
cCblP-dependent.
with
magnesium
317
596
enzyme(s)
enzyme(s)
(mixed)
539
PKInh , and
min
mM blg2+
476
rather
a commercial
dependent
10
510
peak
?B)
flistoncs
377 361
by
larger
36/S
327
stimulated
75 mEI ivg2+,
nuclei
+
36
Protein kinase activity [y- 32T’],ZTP/? ml of fraction
32P from
Pro tamine 10 mM big?+ 75 mbl blg2+
significantly
COMMUNICATIONS
which have
bl
Vol.
93, No.
4. 1980
preferences cAblP
in
of
at
to
pla)
cycle
and
BIOCHEMICAL
(Table
1).
proliferative
least
some
The D.G.
liver
authors Gillan
view
BIOPHYSICAL
of
development, 01
important of
In
AND
these
roles
enzymes at
COMMUNICATIONS
the
importance
of
the
synthesis
2nd.
will
critical
RESEARCH
almost points
certainly in
the
calcium
and
activation be
found
grolith-division
cells. IZC~NI~I~LCDI;E~II:N? thank B. Braceland for for preparing the figures.
making
the
[y- 3’ l’].ATP
Costa, 51.) I’uller, D..J.51., lladdock Russell, D., Gcrner, E.K., 1.1977) ) Riochim. Biophys. :1cta, G, 410-426. 2. Phillips, Y.R., Shepharci, r.A., Stein, J.L., Kleinsmi th, L.J., Stein, G.S., (1979)) Biochim. Riophys. Acta, 5(;5, YZb-346. Kleinsmith, L..T., (1975)) in The Cell Nucleus, Vol. 1.1, Ed. 3. liush, H., Academic Press X.1.. ZZZ-761. I\‘hi tfielcl, J.1:. , Boynton, ;\.I~., blncblanus, cJ.I’. , Sikorsha, bl., 4. Tsnng, B.K., (1979)) !101. Cell. Biochem., 2, 155-179. Boynton, A.L., Whitfield, .J.l’. , (1980)) Esp. Cell Rcs., in .5 . press. kish, \...‘I., lileinsmith, IL.J., (1973)) ,J. Hiol. Chem., ?49 0. -3 75n-760. Desjardins, P.R., I,ue, l’.l:., Liew, L.C., (;ornall, A.(;., (197?), 7. CRI‘ . .J. Bioch., 50, 1?49-1’59. Tabcda, ;‘l., i\latsumur-a, S., Naknyn, Y., j19731, .J. Riochem., s. 2, 743-751. 9. Rui.don, R.N., Anderson, S.L., (1972)) Iiiochcm. Biophys. RE..s. Commun. , s, 1499-1508. 10. Thornburg, W., Game, S., c)‘~l~llcy, A.[:. , Lindell, T.,J., (1979), Riochim. Biophys. Acta, 571, X-34. 11. Guidotti, A., Chuang, D.M., IlollenbecX, R., Costa, F.. , (1978)) Nucleotide Res., z, 185-197. ?\d\- . Cyclic 12. blednieks, )I. I. , Jungmann, R.A., Schweppe, J.S. , [ 1979) , Arch. Biochem. Hiophys., 197, 185-19:. 13. Flynn, I .bl., Chappell, ,J.Ii., (1964)) Biochcm. ,I., s, 147-149. 13. !lac?lanus, ,J.P., (1979)) .Anal. Biochcm., E, 107-310. 15. Takai, 1’. , hishimoto, :“\. , Inoue, bl., Nishizuka, Y., (1977)) Chem., s, 7603-7609. .I . Iiiol. 1 h . Dills, W.L., (Yooclwln, Ch.D., Lincoln, T.>1., BC~VO, .J..k., Ke~~lltcl, 1’. J., Corbin, J.D., Krebs, I..(;. , (1979)) Adv. Cyclic Nucleotide Rcs., 10, 199-217. 17. Bradford, bl.bl., (1976)) Anal. Riochcm., 7,1, 248-254. 18. Krehs, G.E., Henvo, G.A. , (1979) , in Ann. Rev. Biochem., g, 9?4-959. Shoji, Kuo, J.F., (1976)) Biochim. Riophys. :ict:l, 19. Ku<), K.N., 4 j 7 142-149. -’ ,,,;,,~~~,~,~‘;l~~,~.1~. , (1976)) .J. Riol. Chem., 751, A783-438h. 20 . IKu0 P.11. , (1979) , Biochim. 21.: ;~‘ A. ) >IBenpkfY, 324-33:. Riophys. Acta, %& 1.
1203
93, No.
April
BIOCHEMICAL
4, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
29, 1980
Marianna Sikorska, F’. Roy Walker and
1196-1203
Animal and Cell l~hysiology Group Division of Biological Sciences Nat ional Research Count i 1 of Canada Ottawa, Canada Kl:l OR6 Received
March
11,
1980
Soluble proteins of rat liver nuclei I\‘ere applied to a DIALScphacel column and eluted \iith a linear gradient of 70-600 mill NaCl. Three major classes of nuclear protein kinase were identified. There Ivcre types I and II c:UIP-dependent protein Ainasc, a Ca-cnlmodul in-dependent protein hinasc, and a third enzyme which 1~31; stimulated by c.\biI’-dcpendcnt protein hinase inhih i tor. ‘The
patterns
protein
of
PK
(protein
phosphor~lation
division
cycle
suggesting
change
[l-3).
th;1t
hinnsc)
There
1'6s
a h-C)
s.
and
i 1’ 3 t e cl n 0 n - n e 0 I) 1 3 s t i c cells
cl\,iIP
surge
,\lorcovcr,
in
deprivation
almost
immedintel!~
fcx
as
cation
requirements
nuclei
(b-lo),
IlLlclcus
ha5
dependent
Cl
cultivated
calcium
,I5
late
example,
can
tlbo
to
need
induced
the
initiation
in
regenerating calcium
in
late
C!l,\lP)
and
a.5 tkelvc
or
t).pe
forms
growth-
evidence of
I)?;.1 liver
and
a brie1
(A’,. Gl
initiate
I,!, calcium,
as man)’
of
DKA synthesis
to
nuclear their
lines
both
arrested
phase
by
DNA synthesis, IT
c.lblP-dependent
of
PK liith
PI<
di f‘fcren
:i n d 5 u h s t r :I t c p r c f c r c II c c 5 h :I v e h I i’ II f 0 II n d i II but
always
the
presence
been
1’6 activity
of
f : 1980 fly .A cadem,c Prec.5, of reproduction in an-v f‘orm
ciZbiP-cleI)clldcIIt
controversial has
Inc. resertped.
FE.5 in
(3,7,9,11).
recently
been
0006-291X/80/081196-08$01.00/0 Cop-vngh All righrs
in
initiate
cells be
role
and
through
several
hcpatocytes
phase liver
pass
also
synthcsi c 111 t
1’11u 5 , for
as cells arc
play
may
activity
1196
found
flolicve in
thymic
the r , cANI’lymphocyte
Vol.
93, No.
nuclei several
4, 1980
1112). types
cAlilP-dependent by
BIOCHEMICAL
In
this
0P Pli Pk:,
Ca-calmodul
AND
BIOPHYSICAL
communication in and
rat
liver
we
report
nuclei,
a hitherto
RESEARCH
the
presence
including
unnoticed
COMMUNICATIONS
trvo
PK which
is
of types
of
stimulablc
in. IlATL:.RIALS
Bovine serum albumin, CAMP, cGblP, calf thymus histones Sigma type 11,2 (mixed) and Sigma type V-S (111) , beef heart protci n kinasc inhibitor (PKInh), salmon sperm protaminc, 3-isobutyl-1 methyl-xanthinc (IBNX) were obtained from Sigma Chemical Co. (St. Loui s , MO, USA) ; DEAL-Seyhacel ~3s obtained from l’harmac ia (Uppsala, Sweden) ; [ 2,8fl]cAMP (.37 mCi/mmole) was obtained from Amershan Corp. (Oahvillc, Ontario, Canada) . [ y- 32 P].A?‘P was prepared according to Flynn and Chappell (13), and calmodulin was purified to homogeneity from rat testis accordj.ng to ~Iacblanus (14)
Isolation -__~-extract.
of
nuclei
and
preparation
of
soluble
nuclear -___
p rotein -
Liver tissue from specific pathogen-free male Sprague-Uawley rats (1’30-110 g) c~as homogenized in b volumes of buffer A (50 mbl J’ris-HCl, 25 mM KCl, 5 mbl blgC12, 250 mi4 sucrose, pfl 7.5) and then centrifuged at 700 gav for 10 min at 7°C. The pellet was washed with buffer A, and resuspended in 5 volumes of buffer A containing 1: Triton ly-1OU. After centrifugation the pcllct icas \\,ashed again with buffer A. The nuclear preparations were slightly contaminated by mitochcndria (about 5”, of total mitochondrinl mononminc osidasc and s:l:-i:ini; dehydrogenasc activity was associated with nuclei), but contained neither lysosomcs (asscsscd hy Eluorescencc microscopy after staining with acridinc orange ) nor mi crosomt‘s (indicated by no NM~Pll cytochromc c rcductasc activity). h’iuclci hiore extracted with (1.h I\1 NaCl in buffer 13 (20 m,\l ‘Pris-ilC:l, 3 mN &Cl z 3 mbl l:l)TA, 50 mb1 ~-mcrcaptoethn~iol , pll 7. 5 j . The nuclear suspension \kas centrifuged for 15 min at 20,000 gav, the pellet lizis discarded, and the supcrnatant diluted lo-fold with buffer H. The precipitate formed in the diluted supcr;latant 1~:1s removed by celltrifugnt ion at 10,000 ~av for 10 min, and the clear supernatant containing SO”, of the original nuclear proteins and about 80’: of the total nuclear PK activity was applied to a III’AtiSephncc\l column, as described in the figure legends. PK
(Protein hinase) assay Pli activity was assa)~cd according to Tahai et al. (1 sj . ?‘hc assay mixture (0.15 ml) contained: 7.5 nmole [y-‘*P]ATP (2 x 10” cpm/nmole), 7.5 pmole Z-mercaptocthanol, 1.25 or IS.75 Umolc 100 pg of mixed histonc, historic 1lp.C 1 2 ) 5 umolc Tris-klC1 (pll 7.51, and the appropriate enzyme fraction. The 111 or ;~rotai.line, mixture was incubated for 5 min at 3O”C, and the react ion was Bovine serum stopped by adding ZO”,, trichloroacetic acid (‘l‘(X). albumin (0. 5 nig) has added a.5 a carrier. The acid-prccipitnblc in 1.0 N Ya1)fl arid matcrinl was dashed three times h) dissolving rc-prccipitnting in TCA. Ihc radioactivity of 321’ incorporated
1197
Vol.
93, No.
into liquid
the
Other
BIOCHEMICAL
4, 1980
protein scintillation
substrates counter.
AND
was
BIOPHYSICAL
measured
RESEARCH
with
COMMUNICATIONS
a Beckman
LS
255
procedures
cAblP-Scpharose was prepared as described by Dills et al. (lb). The coupled density of immobilized N.;“- I12N(CH2) 2-cAb!P was 6 ~molc/ml of scttlcci gel. The regulatory subunit of cAbif’-dependent PK attached to this gel was isolated and purified by elution with 1 ml\1 cGi\lf’, concentration on an Amicon filter (Xb15) and extensive dialysi 5. Protein content was measured by a dye-binding assay (Hio-Rad: 17). RESULTS The classical from
cytoplasmic forms
DEAE-Sephacel
AND DISCUSSIOh’
extracts (I
and by
of
II)
of
100
and
liver
cells
cAMP-dependent 200
mbl NaCl
contained
the
PK,
were
\ihich
respectively
l
x
two eluted
(18).
With PK M,. Withad PK Inh.
,
Figure
1.
The protein kinases from the cytoplasm and nuclei of liver cells. 40 mg of cytoplasmic (A) or nuclear (B) proteins were applied to DEAE-Sephacel columns (0.9 x 30 cm] previously equilibrated with buffer B. The column was washed with the same buffer, and elution was carried out with 100 ml of a linear gradient of NaCl (0. - 600 were collected. mM) , and 2 ml fractions Enzymatic activity was measured with 5 mM Mg'+, mixed histones, 1O-6M CAMP, 2 mMIBMX, and with or without 10 pg of protein kinase inhibitor (PKInh). 1198
The\, ,
Vol.
93, No.
4, 1980
BIOCHEMICAL
phosphorylated
mixed
~crc
inhibited
by
(Fig.
14) -
On the
of
I’K activity
the
same
the
latter,
nuclear PKInh
in
other
hand,
nuclear
was
the
there
extracts, as
inhibited
activity
BIOPHYSICAL
RESEARCH
presence
of
cA)lP-dependent
concentrations were
(Fig.
histones
a specific
in
NaCl
AND
by
eluted
by
5 mbl big*+,
PK inhibitor,
uere
three
two the
COMMUNICATIONS
of
PKInh
distinct
which
peaks
were
eluted
cytoplasmic
enzymes
PKInh
(Fig.
The
300
mFI NaCl
1B). and
was
and
by
and,
third
like
type
stimulated
of by
1B).
. 7orTMNocl*
-70-600
I
mM N&---t 0 x A
* 70 mM NoCl B
Wim PKkh. WitId PK Inh. [3HmhiP-m
. 0
t
x
WhPKhh.
wacd+md cdfT&A Noaddtion
-&....,.I.......,
0
Figure
2.
IO
20 30 40 FRXW+JMJWEFl
50
60
70
DEAE-Sephacel chromatography of soluble liver nuclear protein kinases treated with CAMP. Nuclear proteins (12 mg) were applied to a DEAE-Sephacel column (0.9 x 30 cm) equilibrated with buffer B. After the column was washed, the first elution was carried out with 50 ml of 70 mM NaCl and lo-'M CAMP, followed by the second elution with 100 ml of a linear concentration gradient of NaCl (70 - 600 mM), and 2 ml fractions were collected. To establish CAMP-binding activity, nuclear proteins were loaded on to the column in the presence of 1 nmole of [3H] CAMP (37 mCi/mmole). Before the first elution, the column was washed extensively with buffer PK activity was assayed with 5 mM Mg' , mixed B. 2 mM IBMX, 10m6M CAMP, with or without histone substrate, 10 ug of PKInh (panel A), or (panel B) with 75 mM Mg'+, protamine substrate, with or without 10 ug of PKInh. In panel B, the fractions were also assayed in the presence of 3 x 10e6M calmodulin and 5 x 10W5M Ca2+ (panel B). 1199
Vol.
93, No.
4, 1980
If
8lOCHEMlCAL
the
two
subunits
(181. of
PIis,
Icould
two
them
the
appropriate
altered
by
the
high
On the
other
described
in
inhibitable and
was
the
phosphorylnte two
types
legend
due
of
Fig.
to
of
3)
was
in
m>l ,\lgs+
protein
requirements the three major
to
they
were
extract
them
(1:).
extract
1). on
3:\)
preferred AS
the
PKInh-
of
(Fig.
\k,hich
(Table
(as
peak
70 mM NnCl ts
subunits
1~~ addition
;I ne\i
by
remainelI
cAMP,
hecause
nuclear
C suhuni
5.0
t\io
[R)
(C)
stimulated
generated
eluted
liberated
CAMP-binding
lilagnesium
not
the
to hy
catalytic
used of
due
regulatory
pi-ohably
treatment
COMMUNICATIONS
stimulahle
distinct
active were
which
histones of
two
concentration
cAbIF
were
been
fractions,
NaCl
activity,
probnhly
into
activities
to
hand,
have
identical
their
RESEARCH
activities
should
nearly
However,
cAl\lP
they
dissociate
and
BIOPHYSICAL
PKInh-inhibitable
cAlzlP-dependent which
AND
to ted,
CXPCC
columns
which
TABLE 1 and substrate preferences nuclear classes of Pi;
Protein
Kinasc
activity
[\)
Substrate 5 Fraction His
tone, (type Histone, (type Protamine
mixed IIA, Sigma) Hl V-S, Sigma)
His
tone, (type Histone, (type Protamine
mixed IIA, Sigma) Hl V-S, Sigma)
Histone, (type flistone,
mixed 11.4, Sigma) Hl
(C
subunit
of
(mFl) 75 c.A;ilP-dependent
75
31
31
60 11
51 10
20 8
13 ?
36
(Ca-calmodulin
stimulable
92
-- .7
70
61 108
41 so
26 1’4
25 175
50
(PKInh-stimulablc
illi)
100
81
56
55
108 50
107 64
38 0 I)
‘7 _I 65
expressed as the percentage of that (in 5 m&l magnes iumj as a substrate.
1200
PI<)
PI0
100
I-raction
is
10
concentration 50
10 II
Fraction
PK activity mixed histoncs
hlagnesium 20
assayed
\,ith
Vol.
93. No.
BIOCHEMICAL
4, 1980
AND
BIOPHYSICAL
RESEARCH
TABLE 2 and regulatory (R) activity of catalytic
Effects of cAblP phosphorylating
COMMUNICATIONS
on the subunit.
subunit
(C)
Pbosphorylating activity i-pm 32P from [~-32P]~7E’/100 misci. hi~tones/S nlin
~fi
C subunit* 16),360 R subunit** 1 , ‘00 !I ) 960 (C + R) subunits (C + R) subunits + cAblE’ 15,150 i Extensively dialyzed fraction 10 (Fig. :A) was the source of C subunit ** ITractions ih-46 (Fig. ?A) were poolecl and K subunits were purified on a cA>EP-Sephurose affinity column were
eluted
ivith
purified
(on
activity
of
This
rat
it
the
forms
\\a~
cells
the
FKs
, these
subunits
of
and
hl cNIF.
10-s
fractio.ls
elutcd
The
fir,st
group
36,
and
~3s
second by
FKInh
of
activity
assayed
and
in
activities (‘Table
Two from
I’lis
eluteci
prohahly,
higher Fig.
the
(Fig.
c.UlF
of
2.61) . (‘l’ahlc
2)
non-proIifcr:lting
of
the
were
same
two
gradient
by
higher
hy fraction one
1B.
The
with
protamine
responsible
activity
fractions
was
Although 1201
fractions
the
1,1),
\\hilc
strongly for
and
in
concentrations. fraction the
stimulated the
thirJ
peah
1~3s similar
a suhstratc,
3?-30
C
70 m>l NaCl
in
profile as
putative
peaked
(I:ig.
46,
the
detected KaCl
those
concentrations.
with
mSI NnCl, FKInh
masked
\Vhen
eluted were
b!~ 100-150
h’nC1
revenleJ
enzyme
the
ZH).
of
of
in
in
FKs
range
were
the
was
1,
same
unaffected
75 mbl big?+
nuclei
cM]P-dependent
the
groups
peaked
were
the
c:lblP-dependent
i:igurc
10
r-c-adding
proportions
activities
which
in
by
different
by
seemingly
one,
turn,
further
recluccd
fraction
and
of
eluted
\vas
lihen
Fli.
minor
the
from
in
3.21 anti,
, substantially
cytoplasm
activities
minor
(! i g.
C subunits
contain
llowever
46-57
column)
0 f cAbIT’-dependent
the
m&l i\‘CrCl
overcome,
that
Th c high of
:70
putative
seems
liwr
and
cWP-Scpharosc
reduction
Thus ,
120
but
r~.hen the
lower
in
fractions
46-58
liere
still
Vol.
93, No.
4, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
TABLE 3 nuclear PK(s) from fraction ZI\) by calcium and cnlmodulin
rlctivation (Fig.
of
pmoles
Basal activity + Ca2 + (5 x 10-51\1) + Calmodulin (10-6iV) + ca2 + + calmodulin + + Ca2 + calmodulin !:CTA (5 x 10-4bl)
the
fraction
(Fig.
50 Since
PK
(19-20)
(211,
also
have
the
been
stimulable \ihen
assayed
(eluted with
not
was
80-120
protamine
as
stimulated
by
calmodulin
together 7-
abolished
to
this
are
contain stimulated
strikingly
1787
PKInh of
protamine
as
PKInh-stimulable
substrate
activity
fraction
46
preparation
(Fig.
an
stimulated
by
However,
PKInh
in
been
found
this
unaffected
histones
in
presence
in
study
maximally
50 was the
a cGPIP-
has
fraction
in
:A).
enzyme
the
in
was
stimulated
such
unnoticed
rnbl NaCl), substrate
might
PKInhby
of
which
not
the
nuclei
10m6bI
5,
20,
cGI\IP or
fractions
active
3))
in
and
5 x 10d5
was
75 mb’
calcium
31-40 75 mizl Fig
and
the
10T6
enzyme’s
chelator
EG’IA
3).
from
proliferntively of
Ca-calmodulin
PKs, or
requirements 1202
inactive
the
activities
PKInh, and
and
liver of
which
substrate
2+
strongly
N calcium
increased
specific
classes
magnesium
in
more
1 and
separately)
(Table
main
was
Thus,
while
CAMP,
PK species
(Tables
(but
that
different
with
than
stimulation
by
799
shown).
X-fold
three
3’17
377
Ca-calmodulin.
We conclude cells
834
since
a hitherto
with
activity
in mixed
(data
There
450
cCblP-dependent.
with
magnesium
317
596
enzyme(s)
enzyme(s)
(mixed)
539
PKInh , and
min
mM blg2+
476
rather
a commercial
dependent
10
510
peak
?B)
flistoncs
377 361
by
larger
36/S
327
stimulated
75 mEI ivg2+,
nuclei
+
36
Protein kinase activity [y- 32T’],ZTP/? ml of fraction
32P from
Pro tamine 10 mM big?+ 75 mbl blg2+
significantly
COMMUNICATIONS
which have
bl
Vol.
93, No.
4. 1980
preferences cAblP
in
of
at
to
pla)
cycle
and
BIOCHEMICAL
(Table
1).
proliferative
least
some
The D.G.
liver
authors Gillan
view
BIOPHYSICAL
of
development, 01
important of
In
AND
these
roles
enzymes at
COMMUNICATIONS
the
importance
of
the
synthesis
2nd.
will
critical
RESEARCH
almost points
certainly in
the
calcium
and
activation be
found
grolith-division
cells. IZC~NI~I~LCDI;E~II:N? thank B. Braceland for for preparing the figures.
making
the
[y- 3’ l’].ATP
Costa, 51.) I’uller, D..J.51., lladdock Russell, D., Gcrner, E.K., 1.1977) ) Riochim. Biophys. :1cta, G, 410-426. 2. Phillips, Y.R., Shepharci, r.A., Stein, J.L., Kleinsmi th, L.J., Stein, G.S., (1979)) Biochim. Riophys. Acta, 5(;5, YZb-346. Kleinsmith, L..T., (1975)) in The Cell Nucleus, Vol. 1.1, Ed. 3. liush, H., Academic Press X.1.. ZZZ-761. I\‘hi tfielcl, J.1:. , Boynton, ;\.I~., blncblanus, cJ.I’. , Sikorsha, bl., 4. Tsnng, B.K., (1979)) !101. Cell. Biochem., 2, 155-179. Boynton, A.L., Whitfield, .J.l’. , (1980)) Esp. Cell Rcs., in .5 . press. kish, \...‘I., lileinsmith, IL.J., (1973)) ,J. Hiol. Chem., ?49 0. -3 75n-760. Desjardins, P.R., I,ue, l’.l:., Liew, L.C., (;ornall, A.(;., (197?), 7. CRI‘ . .J. Bioch., 50, 1?49-1’59. Tabcda, ;‘l., i\latsumur-a, S., Naknyn, Y., j19731, .J. Riochem., s. 2, 743-751. 9. Rui.don, R.N., Anderson, S.L., (1972)) Iiiochcm. Biophys. RE..s. Commun. , s, 1499-1508. 10. Thornburg, W., Game, S., c)‘~l~llcy, A.[:. , Lindell, T.,J., (1979), Riochim. Biophys. Acta, 571, X-34. 11. Guidotti, A., Chuang, D.M., IlollenbecX, R., Costa, F.. , (1978)) Nucleotide Res., z, 185-197. ?\d\- . Cyclic 12. blednieks, )I. I. , Jungmann, R.A., Schweppe, J.S. , [ 1979) , Arch. Biochem. Hiophys., 197, 185-19:. 13. Flynn, I .bl., Chappell, ,J.Ii., (1964)) Biochcm. ,I., s, 147-149. 13. !lac?lanus, ,J.P., (1979)) .Anal. Biochcm., E, 107-310. 15. Takai, 1’. , hishimoto, :“\. , Inoue, bl., Nishizuka, Y., (1977)) Chem., s, 7603-7609. .I . Iiiol. 1 h . Dills, W.L., (Yooclwln, Ch.D., Lincoln, T.>1., BC~VO, .J..k., Ke~~lltcl, 1’. J., Corbin, J.D., Krebs, I..(;. , (1979)) Adv. Cyclic Nucleotide Rcs., 10, 199-217. 17. Bradford, bl.bl., (1976)) Anal. Riochcm., 7,1, 248-254. 18. Krehs, G.E., Henvo, G.A. , (1979) , in Ann. Rev. Biochem., g, 9?4-959. Shoji, Kuo, J.F., (1976)) Biochim. Riophys. :ict:l, 19. Ku<), K.N., 4 j 7 142-149. -’ ,,,;,,~~~,~,~‘;l~~,~.1~. , (1976)) .J. Riol. Chem., 751, A783-438h. 20 . IKu0 P.11. , (1979) , Biochim. 21.: ;~‘ A. ) >IBenpkfY, 324-33:. Riophys. Acta, %& 1.
1203