Vol. 178, No. 1, 1991 July 15, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 138-144
GTP-BINDING MEMBRANE PROTEINS IN ACTIVATED AND DIFFERENTIATING T CELLS Tiina Pessa-Morikawa#, Tommy Nordstrijm, Tomas Mustelin, and Leif C. Andersson Department of Pathology, University of Helsinki Haartmaninkatu 3, SF-00290 Helsinki, Finland Received
May 28,
1991
We have earlier reported changes in the GTP binding of several membrane proteins including Gsa and Gia during thymic differentiation of T cells. Using an [a-32P]GTPphotoaffinity labeling technique we have studied the pattern of GTP binding proteins in activated and resting T lymphocytes and in T cells induced to differentiate by TPA. The GTP binding proteins in mitogen-activated T cells resembled those seen in leukemia T cell lines. Treatment of Jurkat, but not of CCRF-CEM, T cells with TPA caused increased GTP-labeling of a 34 kDa protein and Gia. The GTP labeling pattern in TPA-treated Jurkat cells resembled that in resting T lymphocytes. TPA induced de ~DZIOexpression of functional TCR/CD3 on CCRF-CEM and downregulation of TCR/CD3 on Jurkat cells but these changes did not correlate with the altered GTP-labeling patterns. 0 1991 AcademicPi-ess, 1°C.
GTP-binding participate
regulatory
in the transduction
(G) proteins of various
cellular
include Gs and Gi, involved eg. in hormonal and rab-related traffic
and
intracellular
G proteins are implied secretion
phospholipase
cell antigen receptor/CD3 G proteins terminates
[3,4]. Many
comprise
a large family
of proteins
signals [l]. The best known
that
G proteins
control of adenylate cyclase [Z]. The small ras-
in the regulation calcium
C via G proteins
of cell growth,
mobilizing
receptors
intracellular apparently
vesicle activate
[2], and this has also been suggested for the T
complex (reviewed in [5]).
are active
when
their action towards
32P]GTP by uv-irradiation
would
binding
GTP and their intrinsic
the target [1,2]. Therefore be expected to primarily
have used this technique to show that during T lymphocyte subunits of Gs and Gi, together with three unidentified
covalent visualize maturation
GTPase
cross-linking
of [a-
active G proteins.
the adenylate
proteins, ~26, cyclase system
matures and the cells squire immunocompetence.
Others have also reported differentiation-
related changes in the expression
of G proteins
function of the corresponding
or properties
signal transduction
concomitantly
with altered
systems [7-lo]. Here we study the effect of
# To whom correspondence should be addressed. Abbreviations PKC, protein kinase C; TCR, T cell antigen receptor; TPA, tetradecanoyl phorbol acetate. 0006-291X/91 Copyright AN rights
$1.50
0 I991 by Academic Press. Inc. of reproduction in any form reserved.
138
We
in the thymus the a
membrane GTP-binding
p30 and ~34, increase their affinity for GTP [6]. In parallel,
activity
Vol.
178,
No.
1, 1991
mitogen-induced
BIOCHEMICAL
activation
AND
BIOPHYSICAL
and TPA-induced
differentiation
RESEARCH
COMMUNICATIONS
on GTP-binding
proteins
in
membranes of T cells. MATERIALS
AND METHODS
Reaeents [a-32P]GTP (40OCi/mmol) was from Amersham International, calf intestinal alkaline phosphatase from Boehringer Mannheim and other reagents from Sigma. Rabbit antisera against Gia-2 (AS/7) and Gia-3 (EC/Z) were from New England Nuclear, OKT3 and OKT4 from Ortho Diagnostics. m All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum. Human mononuclear cells were isolated from the blood of healthy donors by Ficoll Isopaque gradient centrifugation and T lymphocytes were enriched by passage through nylon wool. T blasts were obtained by stimulating T lymphocytes (1.5 x 106/ml) with 5 Kg/ml PHA for 3-4 days and enriched by Percoll gradient centrifugation. Blood granulocytes were isolated from the erythrocyte and granulocyte-containing pellet after the Ficoll Isopaque centrifugation by lysis of the erythrocytes. Blood monocytes were isolated by Percoll gradient centrifugation. e vroteins : The cells were disrupted by sonication in hypotonic buffer (10 mM Tris, pH 7.6,0.5 n&l MgClL 1 mM EGTA, 1 mM EDTA, 1 n-&l dithiothreitol, 0.1 mM PMSF and 10 l.rgg/ml aprotinin, leupeptin and pepstatin) and membranes were isolated as described [6]. The resulting membrane pellet was suspended in the same buffer and stored at -7BC until use. UV-affinity labeling of membrane proteins (40 pg) with [a-32P]GTP was performed as described [6], except that the 10 min preincubation was performed at room temperature. To remove free [cx-~~P]GTP the samples were either passed through 0.5 ml Sephadex G-25 columns [6] or the proteins were precipitated with 20% TCA and the precipitates washed with diethyl ether. The samples were run by SDS-PAGE (12% gels) under reducing conditions. The gels were subjected to autoradiography or alternatively, the proteins were transferred to nitrocellulose filters and the filters exposed to film. Phosphatase treatment : Membranes were incubated with calf intestinal alkaline phosphatase (10 U/ml) and 100 PM ZnCl2 in sonication buffer at +30°C for 30 min and collected by centrifugation at 100 000 g for 45 min. Calcium measurements and FACS-anaIvsis : [Ca2+li was measured as described [11,12] using the fluorescent calcium indicator Fura-2. Cell phenotype was analyzed by indirect immunofluorescence and flow cytometry using the monoclonal antibodies OKT3, OKT4 and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. RESULTS To compare the repertoire cell types, we separated poIyacrylamide malignant
proteins in T lymphocytes
[a-32P]GTP-photoaffinity
gel electrophoresis.
the band patterns
of GTP-binding
labeled
These experiments
and in the intensity
of individual
membrane
to that in other
proteins
by SDS
revealed marked differences, both in bands, between
cell types (Fig. 1). Some bands, such as a 40 kDa protein
various
(identified
normal
or
as Gia in T
cells 1611,.were seen in all samples, while others displayed certain cell type specificity. GTPlabeled bands similar in size to T lymphocyte while
p26 and p30 were present in most cell lines,
p34 was seen only in T cells, Raji (lane d) and HL-60
patterns were reproducible In comparison
in more than 3 separate experiments
with resting blood T lymphocytes
(~34) band were significantly pattern of GTP-binding
lower in mitogen-activated
(lane h). The GTP-labeling (for each cell type).
the labeling of Gia and a 34-36 kDa T cells (Fig. 1. lanes b and cl. The
proteins seen in T blast membranes resembled that seen in Jurkat and
CCRF-CEM T leukemia cell membranes
(Fig. 2 cl and in membranes
cytes161. 139
from immature
thymo-
Vol.
178, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
kDa - 46
- 30
- 14 abcdefghi Fiaure 1. GTE-photoaffinity labeling of membranes from a) Jurkat T cells, b) blood T cells, c) PHA-stimulated T blasts, d) Raji B cells, e) blood monocytes, f) U937 monocytic cells, g) blood granulocytes, ht HL-60 promyeloid cells and i) K562 erythroleukemia cells.
A.
8.
CCRF-CEM
JURKAT Nq. Control Untreated
TPA-treated
Fluorescence
OKT 3
OKT 3
I min
Cl
kDa
kDa
+
TPA
+
TPA
Figure ZEffects of TEA-treatment (16 nM, 5 d) on the expression of CD3 (a), mobilization of calcium induced by OKT3 fb), GTEphotoaffinity labeling of membranes (cj in CCRFCEM (A) and Jurkat cells (B).
140
Vol.
178, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
One of the bands present in all samples was a heavily labeled 49-52 kDa (p49) band (overexposed
in the shown autoradiograms).
Its intensity
rate of the cells. Resting blood T lymphocytes, bands than the rapidly growing samples of mitogen-activated
correlated
granulocytes
with the proliferation
and monocytes had weaker p49
tumor cell lines (Fig.1). This correlation
was also evident in
T lymphocytes, in which this band was increased compared
to
resting T cells (lanes b and cl. Treatment TCR/CD3
of CCRF-CEM
cells with 16 nM of TPA induced
de nova expression
their ability to induce a rapid rise in the intracellular In Jurkat cells, which TCR/CDJ
constitutively
decreased during
to TCR/CD3
stimulation
concentration
express TCR/CD3,
TEA-treatment
the surface expression
(Fig. 2 a). In addition,
was lost rapidly (already within
of free calcium (Fig. 2 b).
GTP-photoaffinity
the reactivity of these cells
increased
GTE-photoaffinity
CCRF-
proteins (Fig. 2~). Notably, no
increase in ~26, ~30 or p34 could be detected in five separate experiments In contrast, incubation
(Fig.
(not shown).
labeling of the membranes from untreated and TPA-treated
CEM cells showed no change in their pattern of GTP-binding 10 days of induced differentiation.
of the
minutes [12]) and irreversibly
2 b). The expression of CD4 on both cell lines decreased during TEA-treatment
induced
of
receptors within 5 days (Fig. 2 a). These receptors were functional as indicated by
at any time from 1 to
of Jurkat T cells with 16 nM of TPA
labeling of p34 and a 40 kDa protein (Fig. 2~). Higher
doses of TEA did not increase the labeling further. The 40 kDa band coincided with Gia-2 and Gia-3 detected by immunoblotting. these proteins, Inhibition
however,
(T cells do not express Gia-1 11311.The immunoreactivity
did not increase following
of protein synthesis by addition
induced
increase in GTP-labeling
treatment
with
of 20 pg/ml cycloheximide
of
TPA up to 5 days.
did not block the TPA-
of either protein (data not shown). Antibodies
against Goa
did not react with Jurkat membranes (data not shown). The TPA-induced after addition
increase in GTP-labeling
of p34 and Gia was detectable within
lh
of TEA to Jurkat cells and reached its maximum within 4-6 hours (Fig. 3 A). The
increase was 2.53-fold
as determined
more than six independent
by densitometric
scanning of the autoradiograms
from
experiments
and it remained on this higher level for several days.
After removal of TPA the GTP-labeling
of p34 and Gia declined slowly and reverted to the
basal level within
seven days (Fig. 3 B.).
Treatment
with other activators of protein kinase C, such as phorbol
mezerein, also caused an increase in GTP-labeling of phorbol
dibutyrate
3B). To determine
on GTP-labeling
whether
dibutyrate
of p34 and Gia (data not shown).
were treated
with
calf intestinal
The effect
was reversed more rapidly than the effect of TPA (Fig.
direct phosphorylation
of p34 by protein kinase C might cause its
apparent increased affinity for GTE in Jurkat cells, membranes from TPA-induced significant
and
phosphatase
prior
to GTP-photoaffinity
Jurkat cells labeling.
No
change in the labeling intensity of p34 was detected. Instead, a striking increase in
the GTE-labeling
of two smaller bands, 18-19 kDa and 20-21 kDa, occured (Fig 4). DISCUSSION
A number of reports have suggested that a G protein would serve as a signal coupling agent between the TCR/CW
complex and phospholipase 141
C (reviewed
in [5]). However,
this
Vol.
178,
No.
BIOCHEMICAL
1, 1991
A
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
p40 p34
-
LDa
100 24 6 Time after (hours)
12 135 TPA addition (days)
46
4 p40
'PM
-
TPA
-
PdBu
4 p20 4 ~18 14.3
2
03
4
Time after (days)
-
6
drug
--+
04
removal
-
+
-
+
WA
+
Phosphatase
Figure 3.A. Increase in GTE-photoaffinity labeling of p34 and p40 (Gia) in Jurkat cells treated with TEA for various times. The labeling was quantitated by densitometric scanning of autoradiograms and the shown date represent means and SD (where shown) from 3-6 independent experiments. B. Reversal of the TPA- or phorbol dibutyrate (PdBu)-induced change in GTE-labeling of both ~34 and p40. Cells treated with TEA for 4h, washed and cultured without TEA. The shown data represents densitometric scans from a single experiment. Figure 4. Effects of phosphatase treatment membrane proteins from Jurkat cells.
putative
G protein has remained
indirect. Although GTP analogue
activation
GTP$
unidentified
on
GTP photoaffinity
the
and the indications
of a G protein by aluminium
prove that this G protein is functionally muscarinic acetylcholine
inositol phospholipid
labeling
technique
at least ten different
proteins
binding
can be seen [6]. If any of these is a G protein
TCR/CD3,
it should respond to triggering
GTP. We have, however,
remained
been unable
During short time (l-15min)
G proteins
and
from resting
GTP with high affinity and
functionally
associated with the
of this receptor by rapidly increasing its affinity for to detect any such changes stimulation
lectins or anti-CD3 antibodies
unchanged
to visualize
proteins and study changes in their activity. In membranes
human T lymphocytes
with mitogenic
[17].
[18-201.
specificity
stimulation.
turnover
G proteins, such as Gia, does not impair signalling
We have used a GTP-photoaffinity other GTP-binding
complex. In fact, when a
receptor, which is known to use a G protein for signal transduction,
of pertussis toxin-sensitive
through the TCRKM
or the non-hydrolyzable
in T cells (14-161, this does not
linked to the TCR/CD3
was transfected into T cells, it was capable of inducing Inactivation
of its existence are rather
fluoride
results in inositol phosphate formation
labeling of
the GTP-labeling
(data not shown), and there were 142
until
3 days after
of T lymphocytes or isolated membranes
no
of T cell membrane
proteins
membrane
GTPase
changes
in
Vol.
178,
BIOCHEMICAL
No. 1, 1991
activity (unpublished binding
observation).
AND BIOPHYSICAL
Even if all known
domains we cannot rule out the possibility To further
signalling
investigate
the possible
TCR/CD3-negative. TCR/CD3
G proteins
GTP-
role of GTP-binding
TCR/CD3
lines CCRF-CEM
complexes,
TPA treatment has opposite
proteins
while
in TCR/CD3
and Jurkat. The latter the former
effects on the expression
normally
is
and function
of
on these cells. TPA induces expression of functional TCR/CD3 complexes on CCRF-
CEM, while
it blocks the function
of these receptors on Jurkat cells. In neither
however,
was there any correlation
between
membrane
proteins. Both CCRF-CEM
and Jurkat contained
~34, and no induction functional
TCR/CD3
of these proteins
functional
receptors
occured in CCRF-CEM despite the appearance
for direct phosphorylation phosphatase
system related to mature T cell physiology.
remains unclear. The effects of TPA, phorbol dibutyrate
are likely to involve PKC-mediated
phosphorylation.
of ~34 by in vitro of membrane
vitro increases their affinity for GTP severalfold. affected by ma-treatment.
Thus, in vim
The
and mezerein find evidence
treatments prior to GTP-labeling.
proteins
samples.
and, at least partly, inactivated
proteins via phosphorylation
We did not, however,
phosphatase
of two small GTP-binding
treatment
phosphorylated
Jurkat cells correlates with a
arrest of these cells. Increased labeling with GTP might reflect
of a signal transduction
the labeling
of
of p34 (and
loss of TCR/CD3 function.
mature phenotype and growth mechanism of induction
of
very low levels of ~26, ~30 and
The presence of p34 in resting T cells and TPA-induced functional maturation
cell line,
and GTP-labeling
complexes. In Jurkat cells, on the other hand, GTP-binding
Gia) was induced following
Instead,
have very similar
that some of them escape detection.
we used the two human T cell leukemia
expresses high levels of functional
RESEARCH COMMUNICATIONS
increased
markedly
these proteins
during
apparently
are
and removal of the phosphate
There are precedents
[21,221. The in tivo phosphorylation
for regulation
in
of G
of these proteins was not
The identity of these proteins remains unknown,
but they might
belong to the the ras or rub protein families. Recent findings indicate that an early tyrosine phosphorylation required
for activation
apparently
of phospholipase
protein. Although TCR/CDJ
C in T ceils (23, 241. The TCR/CD3
activates a tyrosine kinase, most likely pp59frn and/or
might direcly phosphorylate
phospholipase
they are compatible
complex
pp561Ck, which, in turn,
C on tyrosine 1251 without
our results do not rule out the possibility
signalling,
event precedes and is
involvement
that a G protein
of a G
is involved
in
with such a model.
ACKNOWLEDGMENTS We wish to thank MS Monica Academy of Science, the Magnus
Schoultz
Ehrnrooths
Perklens Stiftelse, the Finska Likaresgllskapet
for technical
Foundation,
assistance and the Finnish
the Finnish
Cancer Society, the
for financial support.
REFERENCES 1. 2.
Boume, H.R., Sanders, D.A. and McGormick, F. (1990) Nature 348,125-132. Bimbaumer, L., Abrahamowitz, J. and Brown, A.M. (1990) Biochim. Biophys. Acta
3.
Barbacid, M. (1987) Ann. Rev. Biochem. 56, 779-827.
1031,163-224.
143
Vol.
178,
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Balch, W.E. (1990) Trends B&hem. Sci. 15,473-477. Altman, A., Coggeshall, K.M. and Mustelin, T. (1990) Adv. Immunol. 48,227-360. Pessa-Morikawa, T., Mustelin, T. and Andersson, L.C. (1990) J. Immunol. 144,26902695. Lamer, A.C. and Ross, E.M. (1981) J. Biol. Chem. 256, 9551-9557. Luetje, C.W., Gierschik, I’., Milligan, G., Unson, C., Spiegel, A. and Nathanson, N.M. (1987) Biochemistry 26,4876-4884. Mullaney, I., Magee, AI., Unson, C.G. and Milligan, G. (1988) Biochem. J. 256,649656. Daniel-Issakani, S., Spiegel, A.M. and Strulovici, B. (1989) J. Biol. Chem. 264, 2024020247. Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) Nature 295,68-70. Nordstrom, T., Stahls, A., Pessa-Morikawa, T., Mustelin, T. and Andersson, L.C. (1990) B&hem. Biophys. Res. Comm. 173,396-400. Kim, S., Ang, S.-L., Bloch, D.B., Bloch, K.D., Kawahara, Y., Tolman, C., Lee, R., Seidman, J.G. and Neer, E.J. (1988) Proc. NatI. Acad. Sci. USA 85,4153-4157. Sasaki, T. and Hasegawa-Sasaki, H. (1987) FEBS Lett. 218, 87-92. O’Shea, J.J., UrdahI, K.B., Luong, H.T., Chused, T.M., Samelson, L.E. and KIausner, R.D. (1987) J. Immunol. 139,3463-3469. Sommermeyer, H., Schwinzer, R., Kaever, V., Behl, B. and Resch, K. (1990) Eur. J. Immunol. 20,1881-1886. Koretzky, G.A., Picus, J., Thomas, M.L. and Weiss, A. (1990) Nature, 346,66-68. Gelfand, E.W., Mills, G.B., Cheung, R.K., Lee, J.W.W. and Grinstein, S. (1987). Immunol. Rev. 95,59-87. Mustelin, T., Pessa, T., Lapinjoki, SE’., Gynther, J., JIrvinen, T., Eloranta, T. and Andersson, L.C. (1989) Adv.Exp. Med. Biol. 250,301-303. Chaffin, K.E., Beals, C.R., Wilkie, T.M., Forbush, K.A., Simon, M.I. and Perlmutter, R.M. (1990) EMBO J. 9,3821-3829. Pyne, N.J., Murphy, G.J., MiIIigan, G. and Houslay, M.D. (1989) FEBS lett. 243, 77-82. Lapetina, E.G., Lacal, J.C., Reep, B.R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. USA 86,3131-3134. Mustelin, T., Coggeshall, K.M., Isakov, N. and Altman, A. (1990) Science 247, 15841587. June, C.H., Fletcher, MC., Ledbetter, J.A., Schieven, G.L., Siegel, J.N., Phillips, A.F. and Samelson, L.E. (1990) Proc. Natl. Acad. Sci. USA 87, 7722-7726. Wahl, M.I., Daniel, T.0 and Carpenter, G. (1988) Science 241, 968-971.
144
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 138-144
GTP-BINDING MEMBRANE PROTEINS IN ACTIVATED AND DIFFERENTIATING T CELLS Tiina Pessa-Morikawa#, Tommy Nordstrijm, Tomas Mustelin, and Leif C. Andersson Department of Pathology, University of Helsinki Haartmaninkatu 3, SF-00290 Helsinki, Finland Received
May 28,
1991
We have earlier reported changes in the GTP binding of several membrane proteins including Gsa and Gia during thymic differentiation of T cells. Using an [a-32P]GTPphotoaffinity labeling technique we have studied the pattern of GTP binding proteins in activated and resting T lymphocytes and in T cells induced to differentiate by TPA. The GTP binding proteins in mitogen-activated T cells resembled those seen in leukemia T cell lines. Treatment of Jurkat, but not of CCRF-CEM, T cells with TPA caused increased GTP-labeling of a 34 kDa protein and Gia. The GTP labeling pattern in TPA-treated Jurkat cells resembled that in resting T lymphocytes. TPA induced de ~DZIOexpression of functional TCR/CD3 on CCRF-CEM and downregulation of TCR/CD3 on Jurkat cells but these changes did not correlate with the altered GTP-labeling patterns. 0 1991 AcademicPi-ess, 1°C.
GTP-binding participate
regulatory
in the transduction
(G) proteins of various
cellular
include Gs and Gi, involved eg. in hormonal and rab-related traffic
and
intracellular
G proteins are implied secretion
phospholipase
cell antigen receptor/CD3 G proteins terminates
[3,4]. Many
comprise
a large family
of proteins
signals [l]. The best known
that
G proteins
control of adenylate cyclase [Z]. The small ras-
in the regulation calcium
C via G proteins
of cell growth,
mobilizing
receptors
intracellular apparently
vesicle activate
[2], and this has also been suggested for the T
complex (reviewed in [5]).
are active
when
their action towards
32P]GTP by uv-irradiation
would
binding
GTP and their intrinsic
the target [1,2]. Therefore be expected to primarily
have used this technique to show that during T lymphocyte subunits of Gs and Gi, together with three unidentified
covalent visualize maturation
GTPase
cross-linking
of [a-
active G proteins.
the adenylate
proteins, ~26, cyclase system
matures and the cells squire immunocompetence.
Others have also reported differentiation-
related changes in the expression
of G proteins
function of the corresponding
or properties
signal transduction
concomitantly
with altered
systems [7-lo]. Here we study the effect of
# To whom correspondence should be addressed. Abbreviations PKC, protein kinase C; TCR, T cell antigen receptor; TPA, tetradecanoyl phorbol acetate. 0006-291X/91 Copyright AN rights
$1.50
0 I991 by Academic Press. Inc. of reproduction in any form reserved.
138
We
in the thymus the a
membrane GTP-binding
p30 and ~34, increase their affinity for GTP [6]. In parallel,
activity
Vol.
178,
No.
1, 1991
mitogen-induced
BIOCHEMICAL
activation
AND
BIOPHYSICAL
and TPA-induced
differentiation
RESEARCH
COMMUNICATIONS
on GTP-binding
proteins
in
membranes of T cells. MATERIALS
AND METHODS
Reaeents [a-32P]GTP (40OCi/mmol) was from Amersham International, calf intestinal alkaline phosphatase from Boehringer Mannheim and other reagents from Sigma. Rabbit antisera against Gia-2 (AS/7) and Gia-3 (EC/Z) were from New England Nuclear, OKT3 and OKT4 from Ortho Diagnostics. m All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum. Human mononuclear cells were isolated from the blood of healthy donors by Ficoll Isopaque gradient centrifugation and T lymphocytes were enriched by passage through nylon wool. T blasts were obtained by stimulating T lymphocytes (1.5 x 106/ml) with 5 Kg/ml PHA for 3-4 days and enriched by Percoll gradient centrifugation. Blood granulocytes were isolated from the erythrocyte and granulocyte-containing pellet after the Ficoll Isopaque centrifugation by lysis of the erythrocytes. Blood monocytes were isolated by Percoll gradient centrifugation. e vroteins : The cells were disrupted by sonication in hypotonic buffer (10 mM Tris, pH 7.6,0.5 n&l MgClL 1 mM EGTA, 1 mM EDTA, 1 n-&l dithiothreitol, 0.1 mM PMSF and 10 l.rgg/ml aprotinin, leupeptin and pepstatin) and membranes were isolated as described [6]. The resulting membrane pellet was suspended in the same buffer and stored at -7BC until use. UV-affinity labeling of membrane proteins (40 pg) with [a-32P]GTP was performed as described [6], except that the 10 min preincubation was performed at room temperature. To remove free [cx-~~P]GTP the samples were either passed through 0.5 ml Sephadex G-25 columns [6] or the proteins were precipitated with 20% TCA and the precipitates washed with diethyl ether. The samples were run by SDS-PAGE (12% gels) under reducing conditions. The gels were subjected to autoradiography or alternatively, the proteins were transferred to nitrocellulose filters and the filters exposed to film. Phosphatase treatment : Membranes were incubated with calf intestinal alkaline phosphatase (10 U/ml) and 100 PM ZnCl2 in sonication buffer at +30°C for 30 min and collected by centrifugation at 100 000 g for 45 min. Calcium measurements and FACS-anaIvsis : [Ca2+li was measured as described [11,12] using the fluorescent calcium indicator Fura-2. Cell phenotype was analyzed by indirect immunofluorescence and flow cytometry using the monoclonal antibodies OKT3, OKT4 and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. RESULTS To compare the repertoire cell types, we separated poIyacrylamide malignant
proteins in T lymphocytes
[a-32P]GTP-photoaffinity
gel electrophoresis.
the band patterns
of GTP-binding
labeled
These experiments
and in the intensity
of individual
membrane
to that in other
proteins
by SDS
revealed marked differences, both in bands, between
cell types (Fig. 1). Some bands, such as a 40 kDa protein
various
(identified
normal
or
as Gia in T
cells 1611,.were seen in all samples, while others displayed certain cell type specificity. GTPlabeled bands similar in size to T lymphocyte while
p26 and p30 were present in most cell lines,
p34 was seen only in T cells, Raji (lane d) and HL-60
patterns were reproducible In comparison
in more than 3 separate experiments
with resting blood T lymphocytes
(~34) band were significantly pattern of GTP-binding
lower in mitogen-activated
(lane h). The GTP-labeling (for each cell type).
the labeling of Gia and a 34-36 kDa T cells (Fig. 1. lanes b and cl. The
proteins seen in T blast membranes resembled that seen in Jurkat and
CCRF-CEM T leukemia cell membranes
(Fig. 2 cl and in membranes
cytes161. 139
from immature
thymo-
Vol.
178, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
kDa - 46
- 30
- 14 abcdefghi Fiaure 1. GTE-photoaffinity labeling of membranes from a) Jurkat T cells, b) blood T cells, c) PHA-stimulated T blasts, d) Raji B cells, e) blood monocytes, f) U937 monocytic cells, g) blood granulocytes, ht HL-60 promyeloid cells and i) K562 erythroleukemia cells.
A.
8.
CCRF-CEM
JURKAT Nq. Control Untreated
TPA-treated
Fluorescence
OKT 3
OKT 3
I min
Cl
kDa
kDa
+
TPA
+
TPA
Figure ZEffects of TEA-treatment (16 nM, 5 d) on the expression of CD3 (a), mobilization of calcium induced by OKT3 fb), GTEphotoaffinity labeling of membranes (cj in CCRFCEM (A) and Jurkat cells (B).
140
Vol.
178, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
One of the bands present in all samples was a heavily labeled 49-52 kDa (p49) band (overexposed
in the shown autoradiograms).
Its intensity
rate of the cells. Resting blood T lymphocytes, bands than the rapidly growing samples of mitogen-activated
correlated
granulocytes
with the proliferation
and monocytes had weaker p49
tumor cell lines (Fig.1). This correlation
was also evident in
T lymphocytes, in which this band was increased compared
to
resting T cells (lanes b and cl. Treatment TCR/CD3
of CCRF-CEM
cells with 16 nM of TPA induced
de nova expression
their ability to induce a rapid rise in the intracellular In Jurkat cells, which TCR/CDJ
constitutively
decreased during
to TCR/CD3
stimulation
concentration
express TCR/CD3,
TEA-treatment
the surface expression
(Fig. 2 a). In addition,
was lost rapidly (already within
of free calcium (Fig. 2 b).
GTP-photoaffinity
the reactivity of these cells
increased
GTE-photoaffinity
CCRF-
proteins (Fig. 2~). Notably, no
increase in ~26, ~30 or p34 could be detected in five separate experiments In contrast, incubation
(Fig.
(not shown).
labeling of the membranes from untreated and TPA-treated
CEM cells showed no change in their pattern of GTP-binding 10 days of induced differentiation.
of the
minutes [12]) and irreversibly
2 b). The expression of CD4 on both cell lines decreased during TEA-treatment
induced
of
receptors within 5 days (Fig. 2 a). These receptors were functional as indicated by
at any time from 1 to
of Jurkat T cells with 16 nM of TPA
labeling of p34 and a 40 kDa protein (Fig. 2~). Higher
doses of TEA did not increase the labeling further. The 40 kDa band coincided with Gia-2 and Gia-3 detected by immunoblotting. these proteins, Inhibition
however,
(T cells do not express Gia-1 11311.The immunoreactivity
did not increase following
of protein synthesis by addition
induced
increase in GTP-labeling
treatment
with
of 20 pg/ml cycloheximide
of
TPA up to 5 days.
did not block the TPA-
of either protein (data not shown). Antibodies
against Goa
did not react with Jurkat membranes (data not shown). The TPA-induced after addition
increase in GTP-labeling
of p34 and Gia was detectable within
lh
of TEA to Jurkat cells and reached its maximum within 4-6 hours (Fig. 3 A). The
increase was 2.53-fold
as determined
more than six independent
by densitometric
scanning of the autoradiograms
from
experiments
and it remained on this higher level for several days.
After removal of TPA the GTP-labeling
of p34 and Gia declined slowly and reverted to the
basal level within
seven days (Fig. 3 B.).
Treatment
with other activators of protein kinase C, such as phorbol
mezerein, also caused an increase in GTP-labeling of phorbol
dibutyrate
3B). To determine
on GTP-labeling
whether
dibutyrate
of p34 and Gia (data not shown).
were treated
with
calf intestinal
The effect
was reversed more rapidly than the effect of TPA (Fig.
direct phosphorylation
of p34 by protein kinase C might cause its
apparent increased affinity for GTE in Jurkat cells, membranes from TPA-induced significant
and
phosphatase
prior
to GTP-photoaffinity
Jurkat cells labeling.
No
change in the labeling intensity of p34 was detected. Instead, a striking increase in
the GTE-labeling
of two smaller bands, 18-19 kDa and 20-21 kDa, occured (Fig 4). DISCUSSION
A number of reports have suggested that a G protein would serve as a signal coupling agent between the TCR/CW
complex and phospholipase 141
C (reviewed
in [5]). However,
this
Vol.
178,
No.
BIOCHEMICAL
1, 1991
A
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
p40 p34
-
LDa
100 24 6 Time after (hours)
12 135 TPA addition (days)
46
4 p40
'PM
-
TPA
-
PdBu
4 p20 4 ~18 14.3
2
03
4
Time after (days)
-
6
drug
--+
04
removal
-
+
-
+
WA
+
Phosphatase
Figure 3.A. Increase in GTE-photoaffinity labeling of p34 and p40 (Gia) in Jurkat cells treated with TEA for various times. The labeling was quantitated by densitometric scanning of autoradiograms and the shown date represent means and SD (where shown) from 3-6 independent experiments. B. Reversal of the TPA- or phorbol dibutyrate (PdBu)-induced change in GTE-labeling of both ~34 and p40. Cells treated with TEA for 4h, washed and cultured without TEA. The shown data represents densitometric scans from a single experiment. Figure 4. Effects of phosphatase treatment membrane proteins from Jurkat cells.
putative
G protein has remained
indirect. Although GTP analogue
activation
GTP$
unidentified
on
GTP photoaffinity
the
and the indications
of a G protein by aluminium
prove that this G protein is functionally muscarinic acetylcholine
inositol phospholipid
labeling
technique
at least ten different
proteins
binding
can be seen [6]. If any of these is a G protein
TCR/CD3,
it should respond to triggering
GTP. We have, however,
remained
been unable
During short time (l-15min)
G proteins
and
from resting
GTP with high affinity and
functionally
associated with the
of this receptor by rapidly increasing its affinity for to detect any such changes stimulation
lectins or anti-CD3 antibodies
unchanged
to visualize
proteins and study changes in their activity. In membranes
human T lymphocytes
with mitogenic
[17].
[18-201.
specificity
stimulation.
turnover
G proteins, such as Gia, does not impair signalling
We have used a GTP-photoaffinity other GTP-binding
complex. In fact, when a
receptor, which is known to use a G protein for signal transduction,
of pertussis toxin-sensitive
through the TCRKM
or the non-hydrolyzable
in T cells (14-161, this does not
linked to the TCR/CD3
was transfected into T cells, it was capable of inducing Inactivation
of its existence are rather
fluoride
results in inositol phosphate formation
labeling of
the GTP-labeling
(data not shown), and there were 142
until
3 days after
of T lymphocytes or isolated membranes
no
of T cell membrane
proteins
membrane
GTPase
changes
in
Vol.
178,
BIOCHEMICAL
No. 1, 1991
activity (unpublished binding
observation).
AND BIOPHYSICAL
Even if all known
domains we cannot rule out the possibility To further
signalling
investigate
the possible
TCR/CD3-negative. TCR/CD3
G proteins
GTP-
role of GTP-binding
TCR/CD3
lines CCRF-CEM
complexes,
TPA treatment has opposite
proteins
while
in TCR/CD3
and Jurkat. The latter the former
effects on the expression
normally
is
and function
of
on these cells. TPA induces expression of functional TCR/CD3 complexes on CCRF-
CEM, while
it blocks the function
of these receptors on Jurkat cells. In neither
however,
was there any correlation
between
membrane
proteins. Both CCRF-CEM
and Jurkat contained
~34, and no induction functional
TCR/CD3
of these proteins
functional
receptors
occured in CCRF-CEM despite the appearance
for direct phosphorylation phosphatase
system related to mature T cell physiology.
remains unclear. The effects of TPA, phorbol dibutyrate
are likely to involve PKC-mediated
phosphorylation.
of ~34 by in vitro of membrane
vitro increases their affinity for GTP severalfold. affected by ma-treatment.
Thus, in vim
The
and mezerein find evidence
treatments prior to GTP-labeling.
proteins
samples.
and, at least partly, inactivated
proteins via phosphorylation
We did not, however,
phosphatase
of two small GTP-binding
treatment
phosphorylated
Jurkat cells correlates with a
arrest of these cells. Increased labeling with GTP might reflect
of a signal transduction
the labeling
of
of p34 (and
loss of TCR/CD3 function.
mature phenotype and growth mechanism of induction
of
very low levels of ~26, ~30 and
The presence of p34 in resting T cells and TPA-induced functional maturation
cell line,
and GTP-labeling
complexes. In Jurkat cells, on the other hand, GTP-binding
Gia) was induced following
Instead,
have very similar
that some of them escape detection.
we used the two human T cell leukemia
expresses high levels of functional
RESEARCH COMMUNICATIONS
increased
markedly
these proteins
during
apparently
are
and removal of the phosphate
There are precedents
[21,221. The in tivo phosphorylation
for regulation
in
of G
of these proteins was not
The identity of these proteins remains unknown,
but they might
belong to the the ras or rub protein families. Recent findings indicate that an early tyrosine phosphorylation required
for activation
apparently
of phospholipase
protein. Although TCR/CDJ
C in T ceils (23, 241. The TCR/CD3
activates a tyrosine kinase, most likely pp59frn and/or
might direcly phosphorylate
phospholipase
they are compatible
complex
pp561Ck, which, in turn,
C on tyrosine 1251 without
our results do not rule out the possibility
signalling,
event precedes and is
involvement
that a G protein
of a G
is involved
in
with such a model.
ACKNOWLEDGMENTS We wish to thank MS Monica Academy of Science, the Magnus
Schoultz
Ehrnrooths
Perklens Stiftelse, the Finska Likaresgllskapet
for technical
Foundation,
assistance and the Finnish
the Finnish
Cancer Society, the
for financial support.
REFERENCES 1. 2.
Boume, H.R., Sanders, D.A. and McGormick, F. (1990) Nature 348,125-132. Bimbaumer, L., Abrahamowitz, J. and Brown, A.M. (1990) Biochim. Biophys. Acta
3.
Barbacid, M. (1987) Ann. Rev. Biochem. 56, 779-827.
1031,163-224.
143
Vol.
178,
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Balch, W.E. (1990) Trends B&hem. Sci. 15,473-477. Altman, A., Coggeshall, K.M. and Mustelin, T. (1990) Adv. Immunol. 48,227-360. Pessa-Morikawa, T., Mustelin, T. and Andersson, L.C. (1990) J. Immunol. 144,26902695. Lamer, A.C. and Ross, E.M. (1981) J. Biol. Chem. 256, 9551-9557. Luetje, C.W., Gierschik, I’., Milligan, G., Unson, C., Spiegel, A. and Nathanson, N.M. (1987) Biochemistry 26,4876-4884. Mullaney, I., Magee, AI., Unson, C.G. and Milligan, G. (1988) Biochem. J. 256,649656. Daniel-Issakani, S., Spiegel, A.M. and Strulovici, B. (1989) J. Biol. Chem. 264, 2024020247. Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) Nature 295,68-70. Nordstrom, T., Stahls, A., Pessa-Morikawa, T., Mustelin, T. and Andersson, L.C. (1990) B&hem. Biophys. Res. Comm. 173,396-400. Kim, S., Ang, S.-L., Bloch, D.B., Bloch, K.D., Kawahara, Y., Tolman, C., Lee, R., Seidman, J.G. and Neer, E.J. (1988) Proc. NatI. Acad. Sci. USA 85,4153-4157. Sasaki, T. and Hasegawa-Sasaki, H. (1987) FEBS Lett. 218, 87-92. O’Shea, J.J., UrdahI, K.B., Luong, H.T., Chused, T.M., Samelson, L.E. and KIausner, R.D. (1987) J. Immunol. 139,3463-3469. Sommermeyer, H., Schwinzer, R., Kaever, V., Behl, B. and Resch, K. (1990) Eur. J. Immunol. 20,1881-1886. Koretzky, G.A., Picus, J., Thomas, M.L. and Weiss, A. (1990) Nature, 346,66-68. Gelfand, E.W., Mills, G.B., Cheung, R.K., Lee, J.W.W. and Grinstein, S. (1987). Immunol. Rev. 95,59-87. Mustelin, T., Pessa, T., Lapinjoki, SE’., Gynther, J., JIrvinen, T., Eloranta, T. and Andersson, L.C. (1989) Adv.Exp. Med. Biol. 250,301-303. Chaffin, K.E., Beals, C.R., Wilkie, T.M., Forbush, K.A., Simon, M.I. and Perlmutter, R.M. (1990) EMBO J. 9,3821-3829. Pyne, N.J., Murphy, G.J., MiIIigan, G. and Houslay, M.D. (1989) FEBS lett. 243, 77-82. Lapetina, E.G., Lacal, J.C., Reep, B.R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. USA 86,3131-3134. Mustelin, T., Coggeshall, K.M., Isakov, N. and Altman, A. (1990) Science 247, 15841587. June, C.H., Fletcher, MC., Ledbetter, J.A., Schieven, G.L., Siegel, J.N., Phillips, A.F. and Samelson, L.E. (1990) Proc. Natl. Acad. Sci. USA 87, 7722-7726. Wahl, M.I., Daniel, T.0 and Carpenter, G. (1988) Science 241, 968-971.
144
