BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REVERSIBLEINHIBITION BY DIAMIDE OF CYCLICAMP-DEPENDENT PROTEINKINASESFROMBOVINETHYROID
Michael McClung and Joan Miller
Division University
of Endocrinology
of Oregon Health Sciences Center Portland,
Received
April
Oregon 97201
26,1977
SUMMARY: The effects of diamide on protein kinases isolated from bovine thyroid were studied. Cyclic AMP-dependent protein kinase activity was directly, rapidly, and reversibly inhibited by diamide. This inhibition was non-competitive with respect to ATP or histone end could be prevented by thiol-reducing agents. However, a cyclic nucleotide-independent thyroid protein kinase was Our data indicate that diamide specifically inhibits protein not affected. kinases which are cyclic AMP-dependent. INZRODUCTION:The cyclic
AMP-protein kinase cascade has been well studied and
is considered to be sn important step in the regulation Yet the functional
TSH (l-5).
by Von Tersch et al. (6) that protein by diamide was of interest.
inhibitedtboth
protein
function by
role of this cascade has not been defined.
Because of our involvement with thyroid
inhibited
of thyroid
kinase activity
protein kinases, the recent observation kinase activity
from swine kidney was
They subsequently reported that diamide and amino acid and glucose uptake in rat
kidney slices (7) and have suggested that diamide might be useful in studies of the physiological
role of protein
Diamide is an oxidizing thiol
kinases in various systems.
agent which has been used to alter
compoundsin several tissues (8-U).
oxidation teins,
of glutathione,
are also oxidized when intact
low concentrations, addition
but thiol
of thiol
it
is non-toxic
It has a high affinity
intracellular for the
groups of other compounds, including procells
are exposed to diamide (11).
and its effect
In
can be reversed by the
reducing agents.
Copyright 0 1977 by Academic Alt rights of reproduction in any
Press, Inc.
form reserved.
910 ISSN
0006291
X
BIOCHEMICAL
Vol. 76, No. 3, I977
AND BIOPHYSICAL
Our study was deeigned to examine the effects kinaees isolated
f-o?? bovine thyroid
diamide specificslag-
inhibits
glands.
the cyclic
RESEARCH COMMUNICATIONS
of diamide on protein
These data demonstrate that
AMP-dependent protein
kinase activity.
MATERIALSANDMETHODS: Preparation of protein kinases: Fresh bovine thyroid glands were homogenized and fractionated by the method of Wolff and Jones (12). All procedures were performed at k" C. The 37,000 X g supernatant was centrifuged at 105,000 X g for 1 h in sn IEC Model B-60 ultracentrifuge. This post7iicrosomal supernatant was dialyzed overnight vs 10 mMTris*Cl, 1 mMdithiot.hreitol, pH 7.2 (buffer A), added to a DEAE-cellulose column (2.5 x 40 cm) equilibrated with buffer A, end the protein kinases were eluted with a linear gradient of NaCl, O-500 mM, in buffer A. Three peaks of protein kinase activity were found. The first two eluted at 50 and 125 mMNaCl, respectively, phosphorylated mixed histones, were stimulated 5-fold by cyclic AMP, pOSBe55edcyclic AMP-binding activity, and seemedidentical to the two histone kinase peaks previously described in bovine thyroid glands (3,4). The third protein kinase peak eluted at 200 mMNaCl, phosphorylated phosvitin but not hietone, snd was cyclic AMP-independent. In several respects, this phosvitin kinase hes been shownto be distinct from both the cyclic AMPdependent kinase holoenzymes and their free catalytic subunits (M. McClung, J. Miller, manuscript in preparation). The histone kinase peak which eluted at 125 mMNaCl (peak II) was pooled and precipitated by adding solid ammonium sulfate (32.5 g/100 ml) with constant stirring. The precipitate was resuspended in 50 mMpotassium phosphate, pH 7.0, containing 2 mMEDNA(buffer B) and then chromatographed on a 2.5 x 90 cm co1""3 of A M agarose (BioRad) equilibrated in buffer B. Histone kinase and [ H]cyc I ic AMP-binding activity elated as a single peak which was devoid of phosvitin kinase activity. This preparation was used in the following experiments unless otherwise stated. Protein kinase assays: Standard kinase assays were performed in triplicate in an incubation volume of 0.1 ml contt@ing 50 mMpotassium phosphate, pH 7.0, 10 mMmagnesiumchloride, 0.1 mM[y P]-ATP (specific activity 200-400 cpm/pmole), 200 M histone II-A, and enzyme. When added, cyclic AMP concentration was 2 @f. After incubation for 20 min at 37' in a shaking water bath, reactions were terminated by spotting 50 & assay mixture onto 2x2 cm squares of Whatman3!#4 filter paper. The filter squares were processed as described by Corbin snd Reimsnn (13). Using purified enzyme preparations, kinase activity wa8 linear with respect to time and enzyme concentration. Phosvitin kinase activity was assayed in a similar manner except the MgCl concentration was 2 mMand 200 a phosvitin was used as substrate instead of ii istone. Gilman's method of measusing f3H]cyclic AMP-binding to protein kinases was employed using 5 pmoles [ HIcyclic AMP (14). Protein concentration was determined with the method of Lowry (15). Diamide, pr@ein kinase ubstrates, and thiol reducing agents were obtained kom Sigma; [y P]ATP and [ $ ]cyclic AMPwere purchased from NewEngland Nuclear. REWLTS :
Diamide markedly inhibited
dependent manner (Fig. absence of cyclic
Inhibition
was observed in both the presence and
AMP, although the effect
was more pronounced. kinase activity
1).
histone kinase in a concentration-
on cyclic
At &amide concentrations
was less than 5% of initial
911
AMP-stimulated activity
of 10 m&land greater,
total
values, and the response to cyclic
BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL
DIAMIOE
RESEARCH COMMUNICATIONS
(mM)
Mgure 1. Effect of diamide concentration on thyroid histone kinase activity measured in the presence (O-4) and absence (O--O) of 2 @I cyclic expressed as pmoles phosphate trsnsferred/mg protein/min AMP. Kinase activity (mean + S.E. of triplicate determinations).
The half-maximal
AMPwas no longer apparent.
inhibitory
concentration
of
diamide waa 0.8 mM.
Changesin substrate concentration A reciprocal
plot of the data
competitive with histone. inhibition
did not alter the effect
(Pig. 2) revealed that the inhibition
was non-
Similar experiments also demonstrated non-competitive
-5 with respect to ATP. The Km for ATP was 1.9 x 10 M in both the
presence and absence of 20 mMdiamide.
Inhibition
by diamide was also observed
when substrates other than the mixed histone fraction l&ion
of diamide.
were used.
The phosphory-
of protamine, casein, and other histone preparati'ons was decreased
approximately
The effect
90%by adding 20 mMdiamide in the presence of cyclic
ofthiol
to diamide was examined.
reducing agents on kinase activity Addition
and on its
of 10 mM2-mercaptoethanol,
912
AMP.
response
dithiolthreitol,
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
s Figure 2. LIneweaver-Burke plot of the relationship between histone concentration and diamiae inhibition of thyroid histone kinase activity in the presence ma absence of 2 fl cyclic AMP. l/S = l/histone concentration (mg/ml). l/V = l/pmoles phosphate tranaferred/mg protein/min. (Q-O) NO additions, (-1 20 mMaiamiae, (O--O) cyclic AMP, (O-4 cyclic AMP + 20 mMaiamiae. Km (hiatone) = 2.7 n&ml.
or reduced glutathione
slightly aufricient
(Table I).
increased hiatone phosphorylation
by kinase only
However, when these agents were added in concentrations
to reduce the diamide in the asaay, no significant
kinase activity
decrease in
was observed.
To study the reversibility
of kinase inhibition
by diamide, purified
histone kinase waa incubated on ice for 15 minutes in 0.5 ml 50 mMpotassium phosphate, 2 mMEIYTA, pH 7.0, with and without dialyzed
overnight
10 mMaiamide.
Each sample was
ve 50 mMpotassium phosphate, 2 mMEIYTA, pH 7.0, and then
assayed for kinase activity.
Even after
dialysis,
activity
was decreased in
the sample which had been pre-incubated with diamide (Table II). kinase activity
addition
in the diamide-treated
of 18 mMaithiothreitol
To exsmine the specificity
sample was fully
However,
restored by the
to the assay medium. with which diamiae inhibits
cyclic
AMP-dependent
protein kinases, each of the three kinase peaks which were eluted from the DEAE
913
BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table I. Effect ofthiol reducing agents on dismide inhibition of protein kinase. Standard histone kinase assay conditions with 2 $4 cyclic AMPwere used. Kinase activity expressed as pmoles phosphate transferred/mg protein/min + SE of triplicate determination.
Protein kinase activity Addition
No dismide
None
534 f 27
208 2 20
641 + 27
614 + 44
655 + 38
626 f 12
680 2 24
626
Dithiothreitol, Glutathione,
10 mM 10 mM
2-mercaptoethanol,
10 mN
5 mhfdiamide
+ 59
Table II. Reversibility of diamide inhibition oC kinase activity following removal of diamide by dialysis. Assays were performed under standard conditions with 2 m cyclic AMPand additions as listed. Results shown are the average f S.E. of triplicate assays expressed as pmoles phosphate transferredfmg proteinrmin.
Kinase activitv ----Additions to kinase assay
Pre-incubation with 10 mMdiamide
Pre-incubation without diamide
None
307 2 21
42 210
Dithiothreitol,
5 mM
351 + 18
283 + 32
Dithiothreitol,
18 mM
317 + l?
322 + 24
column were assayed with and without diamide (Table III). both peaks of cyclic affect
the activity
preferentially [3H]cyclic
AMP-dependent protein kinase. of the cyclic
AMP-binding to either
However, diamide did not
nucleotide-independent
phosphorylates phosvitin
(peak III).
protein kinase which
Dismide did not alter
of the histone kinases.
914
Diamide inhibited
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table III. Specificity of protein kinase inhibition by diamide. Peaks I, II, and III eluted from IEAE-cellulgse column at 50, 125, and 200 mMNaCl, respecsively, as described under Methods. [ H]cyclic AMP-binding is expressed as pmoles [ H]cyclic AMPbound/mg protein. Histone and phosvitin kinase were assayed under standard conditions, and activity is expressed as pmoles phosphate transferred/mg protein/ min. AU assays were performed in the presence and absence of 20 mMdismide, and the results listed are meansof triplicate determinations.
Activity Fraction
and activity
assayed
Without diamide
$ inhibition by dismide
With diamide
Peak I [3H]cyclic AMPbinding Aistone kinase, no cyclic AMP Ristone kinase, 2 @4cyclic AMP
3::; 162.9
2.0 11.0 14.2
6Ro 91
14.0 42.9 246.1
14.7 17.1 28.3
6: 89
224.5
220.1
0
Peak II [ 3~lcyclic AMPbinding Histone kinase, no cyclic AMP Histone kinase, 2 @4cyclic AMP Peak III Phostitin
kinase
DISCWSION: The data in this report confinn the observation that protein kinase activity
is reversibly
shown that this
inhibited
by diamide (6).
occurs with cyclic
et al. (6) did not convincingly AMP-dependent protein by only lo-16%. presence of cyclic
More importantly,
AMP-dependent protein kinases.
we have Von Tersch
establish that they were studying a cyclic
kinase, since addition of cyclic
As evidenced by the Y-fold
increase in enzyme activity
AMPand the associated [%]cyclic
the kinases which could be inhibited
AMP stimulated activity in the
AMP-binding activities,
in our studies were clearly
cyclic
AMP-
dependent.
The effects
observed with diamide differ
from those seen with other protein investigated bition
the inhibition
in several important respects
kinase inhibitors.
of thyroid
Kariya and Field recently
protein kinase by adenosine (16).
was seen only when adenosine or one of its
915
derivatives
Inhi-
was present in
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION ol2TPI
was separated
Cl esterase
inhibitor
@l-antitrypsin
inhibitor,
stable
Thus,
inhibited
very
The extent
of the
proteinases was less
it
selectively
inhibitory
sensitive,
Ficin
and fruit
the inhibition
well
known
is
that
other
proteinases
u2TPI
inhibits
inhibition The biological
inhibitor
at inflammatory the prolonged influenced
determine matory
by blood whether
loci
phase
ti2TPI
and by what
this
is
and papain
different
were
most
(2,
proteinases
o(2TPI is not
yet
from known.
be in the control
In fact,
Beloff
and tissue
and Tokaji
which
inhibitors.
thiol
It
is
interacts
with
the proteinases
mechanism
this
occurs.
is
but
(14).
In contrast,
that
the mechanism
of
Oc2-macroglobulin.
also
of
one of the probable proteihases
(16)
depends
It
proteinases
However,
of the
(15)
of inflammation,
that
sensi-
may suggest
combination.
proteinases
different
Bl
less
proteinases
serine
thiol
cathepsin
considerably
indicating
origins.
among several
stoichiometric
13) and acidic
proper-
and plant
sensitive,
were
not only
(ceruloplasmin
a new serum component.
among thiol
inhibits
from another
of animal
however,
and from
physicochemical
inhibitor
was,
and
mobilities.
gTP1
similar
proteinases
substantially
may
sites.
that
due to a simple
thiol
functionof
of this
that
thiol
selectively by @2TPI is
had very
weights,
serum components
and stem bromelains
a*-macroglobulin like
that
inhibitor
electrophoretic
distinguished
of susceptibility not
d-trypsin
molecular
two other
thiol effect
investigated.
that
role
and from
appears
Such a difference
tive.
of their
further
glycoprotein)
to qTP1.
0(2TPI
III,
inter
by their
of antigenicities
antithrombin
and @'*-heat
is
by the difference
and $-antichymotrypsin
The examination
ties
from C$macroglobulin,
have
on thiol
released indicated
proteinases,
of further
interest
to
released
in the
inflam-
REFERENCES 1. Heimburger, N., Haupt, H. and Schwick, H.G. (1971) Proceedings of the International Research Conference on Proteinase Inhibitors, ~~-1-22, Walter de Gruyter, Berlin-New York. 2. Sasaki,M.,Yamamoto, H., Yamamoto. H. and Iida S. (1974) J. Biochem. -'75 171-177. 3. Kimmel, J.R. and Smith, E.L. (1954) J. Biol. Chem. 207, 515-531. 4. Englund, T.R., King, T.P. and Craig, L.C. (1968) Biochemistry 1, 163-175.
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