Jun 15, 1989 - PHORBOL ESTER INDUCES DIFFERENTIAL MEMBRANE-ASSOCIATION OF. PROTEIN KINASE C SUBSPECIES IN HUMAN PLATELETS.
BIOCHEMICAL
Vol. 161, No. 2, 1989 June 15, 1989
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 556-561
PHORBOL ESTER INDUCES DIFFERENTIAL MEMBRANE-ASSOCIATION PROTEIN KINASE C SUBSPECIES IN HUMAN PLATELETS Agnes Fournier,
Stephen and
School
of
Andrew
Biological
Bedford
J.Hardy,
South
J.
Clark
W.Murray
Sciences,
Park,
Katherine
OF
Flinders
University,
Australia,
5042
Received Hay 3, 1989 S-. Human platelets contained proteins which cross-reacted with antisera specific for brain protein kinase C-a and -p. When platelets were incubated with 12-O-tetradecanoylphorbol13-acetate there was a rapid accumulation of protein kinase C-cx in the particulate fraction associated with a loss of this subspecies from the soluble fraction. No particulate accumulation or soluble loss of protein kinase C-p could be detected when platelets were incubated with the phorbol ester. B 1989Academic Press,Inc. Protein of
kinase
transmembrane
activated hydrolysis phorbol
C (PKC) is
now recognised
signalling
systems
esters
diacylglycerol.
which
act
by agonist-induced or by tumour promoting
as structural
The biological
role
component
The enzyme is
(1).
by diacylglycerol produced of inositol phospholipids
as a major
analogues of
PKC is,
of however,
complicated by the observation that the enzyme consists family of subspecies which are differentially expressed different
cell
brain tissue II and III)
types
(2).
Probably
which contains three which can be separated
chromatography respectively.
the
best
there
characterised
major forms of PKC (types by hydroxyapatite
is I,
and are encoded by the cDNA's y, pl + p2 and Q Other cells express varying proportions of the
major PKC subspecies (3-6). Although it different PKC subspecies have different pathways,
of a in
is
very
little
information
is predicted functions in on this
that the signalling
point.
In a
recent paper Ase et a1.(7) reported that KM3 cells contain predominately PKC-cr and PKC-p2 and that phorbol ester treatment induced a more rapid membrane translocation and down-regulation of PKC-(32 than of PKC-a. It is clearly important to determine 0006-291X/89$1.50 Copyright All rights
0 1989 by Academic Press, ~nc. in any form reserved.
of reproduction
556
Vol. 161, No. 2, 1989
the
generality
report
that
BIOCHEMICAL
of this
observation.
in platelets,
brain PKC-a and PXC-p, the tetradecanoylphorbol-13-acetate translocation
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
which
In the also
contain
present
paper
enzymes
phorbol ester 12-O(TPA) selectively
we
similar
induces
to the
PKC-a.
Materials and Methods . . . if1 atlon of PKC. Rat brain PKC was purified and assayed as dEscri:ed (8). PKC-a and PKC-p were isolated by hydroxyapatite chromatography (9) of brain extracts partially purified by DE52 chromatography. Incubation and extraction of olatelets. Human platelets were isolated, washed and suspended in platelet buffer as described (10). The platelet suspensions (2ml; 2-6 x log cells/ml) were incubated at 37OC for appropriate times with TPA (150nM) or dimethylsulfoxide (0.1%). After incubation, 2ml of double strength sonication buffer (4OmM Tris-HCl, 4mM EDTA, 1OmM EGTA, 0.5M sucrose, 0.02% leupeptin, 8mM PMSF, 20mM pmercaptoethanol, pH 7.5) was added, and the suspension sonicated for 4 x 10 sec. bursts, Sonicates were centrifuged at 100,000 x g for 30 min. at 4OC. The supernatants were collected (soluble fraction) and the pellets sonicated as above in 2ml of sonication buffer containing 0.2% Triton-X 100. After incubation at 2oC for 30 min. the samples were centrifuged as above and the supernatants collected (particulate fractions). Preparation of samples for immunoblottinq. Aliquots (600~11) of soluble and particulate extracts were mixed with 300~1 of solution containing 40% sucrose, 6% sodium dodecyl sulfate (SDS), 20mM Tris-HCl, 5~~1 P-mercaptoethanol, 5~1 1% Bromophenol blue, pH 6.8 and incubated in a boiling water bath for 3 min. The extracts were stored at -2OOC until required. Protein determinations were by the method of Bradford (11) on trichloroacetic acid precipitates of the extracts. Preparation of antibodies. Antibodies were raised in rabbits peptides specific for PKC-a and PKC-p (5) and to a peptide present in the regulatory domain of PKC-a, PKC-8 and PKC-y (12).
to
Lmmunoblottina of olatelet extracts . Samples of the hot SDS extracts were subjected to electrophoresis on 12% SDSpolyacrylamide gels. Proteins were transferred electrophoretically to nitrocellulose paper which was then incubated with 5% bovine serum albumin and 10% horse serum to saturate non-specific binding sites. The nitrocellulose was incubated with antiserum (1:lOO dilution) for 2 h. Sheep antirabbit was used as the secondary antibody and detection carried out with rabbit anti-sheep coupled to alkaline phosphatase. The phosphatase was detected with 4-chloro-3-indolyl phosphate and nitro blue tetrazolium. The blots were scanned using an LKB Ultrascan laser densitometer coupled to an LKB 2220 integrating printer/plotter. The helium-neon laser source provides monochromatic light of 633nm giving good sensitivity of detection of the purple stain. Each band was scanned within the central two thirds of the band, and peak areas calculated in arbitrary units.
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Discussion
and
Results
AND
In these experiments the association of PKC with platelet membranes after treatment with TPA was quantified using polyclonal
antibodies
antibodies
used were raised
peptides previously
for in
PKC-a and PKC-p.
rabbits
using
the
associated with the variable sequence (5). The specificity of the antisera
in experiments mixture
of
separated of the
specific
with
purified
rat
brain
y, p and a subspecies by hydroxyapatite
antisera
a major
antibody antibody
contains
(Figs.1
protein
and 2).
of MW about
SDS extracts
of human platelets
blocked
by inclusion
shown).
Previous
of
the
studies
Each
80,000
was blocked specifically by the was raised. Similarly isolated
PKC-a and PKC-p reacted only with the corresponding Both antisera also reacted with a MW 80,000 protein hot
a
PKC-a and PKC-p
chromatography
recognised
and binding of either peptide to which the
of PKC described was confirmed
PKC which
and with
The
synthetic
and the
appropriate
have
shown that
antiserum. present in
staining
peptide
could
(data
platelets
be
not
contain
PKC
subspecies with elution profiles from hydroxyapatite columns similar to rat brain PKC-a and PKC-p and which cross-react with
a
bc
a
d
e
b
*“I
01
e
f
9
02
h
h
i’j
Figure 1. Specificity of antisera for PKC-a and PKC-8. Purified rat brain PKC (146ng) was subjected to immunoblot analysis using anti-PKC-a (lanes a-d) or anti-PKC-p (lanes e-h) for detection. Staining was done in the absence of competing peptide (lanes a and e) or in the presence of 200 pg of consensus peptide (lanes b and f), PKC-u peptide (lanes d and g) or PKC-P peptide (lanes c and h). The position of PKC is indicated by arrows. of antisera for PKC-a and PKC-p. Figure 2. Specificity Subspecies PKC-a (lanes a-e) and PKC-p (lanes f-j) were isolated from rat brain PKC by hydroxyapatite chromatography as described in Materials and Methods and subjected to irmnunoblot analysis. Detection was with anti-PKC-a (lanes b-e, lane f) or with anti-PKC-p (lane a, lanes g-j). Staining was done in the absence of competing peptide (lanes a,b,f and g) or in the presence of 200 pg of consensus peptide (lanes c and h), PKC-a peptide (lanes e and i) or PKC-p peptide (lanes d and j). The position of PKC is indicated by arrows. 558
Vol. 161, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lncubetion
Figure 3. Effect of TPA on fraction of olatelets. Particulate iractions were various times of incubation detergent extracts assayed Methods).
monoclonal
antibodies
subspecies differences
(13,141. between
been established
to
tiIn6
(mh.)
PKC activity
in the particulate
isolated from platelets after with 150nM TPA (0) or DMSO (01, and for PKC activity (see Materials and
the
corresponding
rabbit
brain
Thus, despite evidence of catalytic PKC-fi from brain and platelet (131,
that
platelets
contain
forms
of
it
has
PKC
immunologically similar to brain PKC-a and PKC-p. As shown in Fig.3, incubation of platelets with TPA caused a rapid association of PKC activity with the particulate fraction.
When analyzed
translocation
of
with
the
specific
PKC-a was observed
could
not
be detected
time
points
studied.
that
there
in
was a linear
purified rat brain peak area measured
the
(Figs.4
particulate
In separate
fraction it
between to
a clear
and 5) while
experiments
relationship
PKC subjected by densitometry
antisera
the
PKC-fi
at any of
the
was established amount of
electrophoresis of the stained
and the blots over
the range 10 to 150 ng protein (data not shown). The purified brain enzyme is a mixture of PKC-a, PKC-p and PKC-Y, so the limit of detection for each of the individual subspecies is therefore amounts
less than 10 ng. It is of PKC-p were translocated
possible, to the
however, particulate
that small fraction
which were below the limits of detection. It is also possible that PKC-p is rapidly translocated and degraded, as reported and therefore does not accumulate in the for KM3 cells (7), However the staining intensity of PKC-p particulate fraction. in the
soluble
fraction
was unchanged
after
a 15 min.
incubation with TPA, whereas staining of soluble reduced to about 50% of control values (data not 559
PKC-a was shown). Taken
Vol.
161,
No.
2, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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71 Ii
0.4r
0.20
so
PO
P2
PS
PlO
pa0
s90’
/lIr!!Ii0
4
8
Time
(min.)
0.02; 0
12
n
Figure 4. Effect of TPA on PKC-a accumulation in particulate fractions of ulatelets. Hot SDS extracts of platelets were subjected to immunoblot analysis with anti-PKC-a. SO and 530, soluble fractions prepared at zero time and after 30 min. incubation with 150nM TPA. PO, P2, P5, PlO and P30, particulate fractions prepared after zero, 2, 5, 10 and 30 min. incubation with 150nM TPA. The same amount of protein (24 ug) was electrophoresed in each track. The position of PKC is indicated by the arrow. of TPA on PKC-a and PKC-0 in the particulate Figure 5. Effect fraction of platelets. Hot SDS extracts were prepared after incubation of platelets for varying times with 15OnM TPA and aliquots ( 3Opg protein) electrophoresed and immunoblotted as described in the Materials and Methods. Detection was with anti-PKC-a (0) or anti-PKC-p and peak (0,. Staining was quantified by laser densitometry areas are given in arbitrary units. Similar results were obtained in two separate experiments.
we believe
together, induces
the
platelets. cells (7).
that
selective This result It
should
these
results
indicate
that
translocation of PKC-a in human is the reverse of that reported be noted,
however,
that
two forms
TPA for
KM3
of PKC-p
have been reported, and KM3 cells contain predominately PKC-fl2. The relative proportions of the ~1 and p2 forms of PKC in platelets
are
not
It will be of cause a different different changes
cell in the
known.
great interest if the same stimulus (TPA) pattern of PKC subspecies translocation types. This PKC proteins,
could be due to minor structural or alternately to differences
membrane composition. In current whether agonists which interact induce
differential
translocation
experiments with surface of
can in in
we are determining receptors also
PKC.
This work was supported by grants from the Acknowledaments. National Health and Medical Research Council and the AntiCancer Foundation of the Universities of South Australia.
References 1.
2.
Nishizuka,Y. Nishizuka,Y.
Science, Nature,
a, m,
305-311, 661-665,
1986. 1988.
Vol. 161, No. 2, 1989
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
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