Vol.
178,
August
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1991
EFFECT OF
INTRACELLULAR
CHANGES ON THE DISTRIBUTION OF TYROSINE-
pH
SERINE/THREONINE-PROTEIN
G. CLARI,
Dipartimento
di
Biomembrane Received
June
KINASE ACTIVITIES
L. BORDIN,
Chimica
1021-1027
Biologica,
de1 Consiglio
AND
IN HUMAN ERYTHROCYTES
G. MARZARO and V. MORET
Universita Nazionale
di
delle
Padova
Ricerche,
and
Centro
Padova,
delle
Italy
1, 1991
SUMMARY: The pH-dependence of the distribution of Tyr-and Ser/Thr-protein kinases between cytosol and membrane in human erythrocytes was investigated. When the internal pH of human erythrocytes is decreased from 8 to 7.3 the membrane-associated Tyr-protein kinase activity markedly increases at expense of the cytosolic counterpart, whereas the membrane-bound and cytosolic casein kinase activity are unaffected. This different response of the two kinase activities to the imposed variation of intracellular pH may explain why the Tyr-phosphorylation of cytoplasmic domain of band 3 results to be much higher in the ghosts from erythrocytes whose internal pH was 7.3 than that in the ghosts from erythrocytes whose internal pH was 8. By contrast, the Ser-phosphorylation of spectrin o-subunit (band 2) and band 3 results to be practically unchanged in the ghosts from the erythrocytes treated at both pH values. 0 1991 Academic PTXL~S,Inc. Various
specific
corresponding distributed have
between
been
and other possible kinase
found
cytosol
of
The results
Tyr-protein
kinase
activity
counterpart
decreases
to
distribution two above presence is markedly
here
the
when
different of[y3-2
the
higher
the
in the
By contrast
the
erythrocytes
treated
8
same ghosts pH from
Ser-phosphorylation at both
the
distribution
values
from are
is
in
membrane the
cytosolic
intracellular
unaffected.
erythrocytes
whose
these
pH in the
the
separately
unchanged
of
a
membrane-associated while
of band
erythrocytes
of
intracellular the
pH
whether
state
practically
isolated
environment,
investigate
By contrast
remains
Tyr-phosphorylation ghosts
erythrocytes,
enhanced,
extent.
the
of human by ionic
when
as
differently
was to
to 7.3,
markedly
activity
intracellular P]ATP,
show that
as well
being
phosphorylation
from
is
of casein-kinase
Concomitantly,
the
decreased
is
study
may influence
regulating
reported
erythrocytes
in vitro
present pH
(l-13)
besides
structures
affected
intracellular thus
kinases
(14-19),
and membrane
The aim of the
activities,
intact
Tyr-protein
phosphatases
to be differently
factors. change
proteins.
Ser/Thr-and
specific
having
incubated 3 cytoplasmic internal the
the
in
the
domain pH was 7.3.
ghosts
from
the
pH values. 0006-291X/91
1021
$1.50
Copyrighr 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
Methods
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Materials
Human erythrocytes were prepared by centrifugation (at 750 x g for 3 min) of fresh blood collected from healthy donors. To minimize contamination by leucocytes and platelets, the packed red cells were washed three times by centrifugation in buffer A (50 mM Hepes pH 8, 1.5 mM MgSO , 90 mM KCl, 48.5 mM Na2S04, 0.1 mM EGTA, 25 pg/ml chloramphenicol, 0.1 mg/m f streptomycin, 12 mM Glucose and 1 mM Adenosine) and discarding the buffy coat and the upper third of packed red cell layer. Packed red cells were divided into 3 samples at a 10% hematocrit: i.e. in the sample 1, 1 ml packed red cells was suspended in 9 ml buffer A pH 8, while in the samples 2 and 3 3 1 ml packed red cells was suspended in 9 ml for 30 min buffer A adjusted to pH 6.9. The three samples were then incubated 1 and 7.3 for at O°C so that the controlled internal pH were 8 for sample both samples 2 and 3. Subsequently the packed cells recovered by centrifugation of samples 1 and 2 were hemolysed in 28 ml hypotonic 5 mM phosphate buffer (pH 8 for sample 1 and respectively pH 7.3 for sample 2) containing 0.02% NaN and 0.03 (PMSF). On the contrary the pat 9 ed cells mM phenylmethylsulphonyl fluoride were resuspended and incubated for further recovered from sample 3 (pH 7.3) 30 min at O°C in 9 ml buffer A adjusted to pH 8.5, in order to obtain again the internal pH 8. These latter cells, recovered by centrifugation, were then hemolysed in 28 ml of the above hypotonic buffer pH 8. separately recovered from hemolysate of The membranes and cytosol were the three above samples by centrifugation (at 20.000 x g for 20 min). The membrane were then washed twice in the same hypotonic lysis buffer containing 30 p Vanadate (pH 8 for samples 1 and 3 and pH 7.3 for sample 2) and twice in 25 mM TRIS-HCl buffer (pH 8 for the sample 1 and 3 and pH 7.3 for sample 2) containing 0.03 mM PMSF, 0.02% NaN3 and 30 pM Vanadate. Endogqqous phoshorylation of membrane proteins in the ghosts in the presence of [y"-'P'] ATP Endogenous Ser-phosphorylation of membrane proteins was tested by incubating the white ghosts (80 ug proteins) at 30 C for 5 min in 125 ul fXa~i~,~ix~~Ze,so~~~? JO0 mM Hepeg-HCl buffer (pH 7.5), l~yk!p~~;~a;;l P] ATP (6.10 cpm/nmol). Endogenous lation of gembrane proteins was assayed under the same conditions followed for Ser-phosphorylation, except that MgC12 was replaced by 5 mM MnCl . Incubation was stopped by addition of 2% SDS and 1% @-mercap z oethanol (final concentration) followed by a 5 min treatment at 100°C as described in (20). 40 g of solubilized membranes were analyzed by 0.1% SDS -10% PAGE essential P y according to (21). as previously described (20). Membrane-associated and cytosolic casein-kinase activities were assayed by incubating 80 pg of white ghosts and, respectively, 50 ul of dialyzed hemolysate supernatant prepared as described in (20), in the presence of 0.65 mg/ml whole casein as substrate, under the conditions above described for the endogenous Ser-phosphorylation of membrane proteins in the isolated ghosts. After 5 min incubation, the reaction was stopped as above described. Half of reaction mixture was analyzed by electrophoresis followed by autoradiography ag2described in (20). P-labelled casein band, identified by autoradiography, was excised from the gels and counted for radioactivity in a liquid scintillation counter. Membrane-bound and cytosolic Tyr-protein kinase activities were assayed by incubating at 30°C for 5 min, 80 pg of white ghosts and, respectively, 50 1 of dialyzed ammonium-sulfate fraction (60% saturation) prepared from rl emolysate supernatant as described in (20), in the presence of 8 pg poly Tyr) 4:1, as substrate, under the (Glu, same conditions followed for the endogenous Tyr-phosphorylation of membrane proteins. The incubation was stopped and half of reaction mixture was analyzed by electrophoresis followed by autoradiography, as previously degsribed in (20). The corresponding diffuse P-Tyr-labelled band, identified by autoradiography, was excised from the alkali-treated gels and counted for radioactivity in a liquid scintillation counter.
1022
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Other
methods; Miscellaneous Internal pH was assayed according to Borseaux (22). Protein content was determined according to Bradford (23). Synthetic polymer poly (Glu, Tyr) 4:l (molecu 2r mass ranging from 20 kDa to 50 kDa) was purchased from Sigma; [ 4 -PI ATP from Amersham International (U.K.) and whole casein was prepared from commercial pow&r (Merck) as previously described (4). Results When the 7.3
internal
a different
Tyr-protein
kinase
Tyr-kinase
activity,
increased,
while
(See
I).
Table whole
the
above
the
two
explain
the
1) observed
distribution
ghosts
internal
pH was 8 and 7.3
presence
of 5 mM Mn2+.
Tyr-and
are
incorporated
poly
(Glu,Tyr)
Membrane
Human with
is
as into
activity
practically a different of
the
incubated
same
extent
(assayed
on by
response
of
pH
patterns
may (Fig.
samples
whose in
the cytosolic and in human erythrocytes
Ser-protein kinase activity (expressed cpm incorporated
4:l)
whole
Cytosol
Membrane
as into
casein) Cytosol
erythrocytes internal
pH 8
1500
3925
9000
36000
2650
2750
8900
35500
Human erythrocytes with
internal
pH 7.3
The Tyr and Ser protein kinase activities were assayed (as described in in the ghosts prepared from 10 ~1 Methods and Materials) in the cytosol and of two samples of packed human erythrocytes having internal pH 8 and pH 7.3 as countslmin incorporated respectively.3Tyr-kinase activity was expressed P-labelled electrophoretic band of Poly (Glu,Tyr) 4~1 under into diffuse conditions described in Methods and Materials. The Ser protein kinase into whole casein under activity is expressed as counts/min incorporated conditions described in Methods and Materials.
1023
be
unaffected
with[@PlATP
on
pH of
to
intracellular
erythrocyte
pH change kinase activities
Tyr-protein kinase activity (expressed
pH 8 to
results
to the
Ser-phosphorylation
respectively,
Effect of intracellular I. membrane-bound Tyr-and Ser-protein
4:1,
kinase
variation
from
TABLE
from
decreased
of pH. Such
isolated
cpm
Tyr)
and cytosol to the
endogenous the
(Glu, is
change
activities
different
poly
lowered
intracellular distribution i.e. the membrane-associated
of casein
membrane
intracellular
kinase when
the
counterpart
between
mentioned
on
cytosolic
the
is
exhibited by the kinase activities:
assayed the
casein)
protein
human erythrocytes
is
and casein
By contrast, added
pH of
response
the
Vol.
178,
No.
tea
01 Pig.1.
The
a'
b
32
P-labelling
erythrocyte
of
Tyrosyl-residues
is
32P-labelled 1
from
ghosts By
in the is
a
clearly
a'
b
b’
from
its
of
alkali-treated
gels, due
partial
of
2) in
the
to
that
the
spectrin
remains
internal
pH 8 (lane
from
while
the
as
phosphorylation and
of confirmed
alkali-treated
internal
whose
is
pH was 7.3
internal
phosphorylation
of
also
unchanged
much
(lane
b'),
pH was 8 (lane
a').
Seryl-residues
a,b). and band 3 the ghosts are incubated in presence of 10 mM Mg 2+ (more in activating the endogenous Ser-casein-kinase). that
ghosts residues
alkali-stability
Tyr-phosphorylation
whose
erythrocytes
2 shows
the seryl
(24,25).
shows
erythrocytes
contrast
(band
phosphorylation
partially
by
practically unchanged in both Such a Ser-phosphorylation
Fig.
COMMUNICATIONS
RESEARCH
CB
@-subunit
analysis
membranes
Fig. the
BIOPHYSICAL
02
the
due to
as indicated
32 P-phosphoaminoacid
ghosts
b’
spectrin
samples
indicated by its disappearance 32 P-labelling of band 3 by
AND
Autoradiograms showing the effect of variation of internal pH of human erythrocytes on the phosphoryl.at\fn of membrane proteins in the ghof$s incubated 5 min. with [r-P] ATP in the presence of5mMMn . The ghosts were prepared from the two samples of human erythrocytes having internal pH 8 (lanes a,a') and 7.3 (lanes b,b') respectively. Lanes a',b' show the alkali-treated gels corresponding to the alkali-untreated gels a,b. Lane CB shows the Coomassie blue-stained gel. Autoradiograms were exposed for 16h. Autoradiograms showing the effect of variation of internal pH in human erythrocytes on the phosphorylation of mpbrane proteins in their ghost?+ incubated for 5 min. with [Y- P] ATP in the presence of Mg (10 mM). The ghosts were prepared from the two samples of human erythrocytes having internal pH 8 (lanes a,a') and 7.3 (lanes b,b'), respectively. Lanes a',b' show the alkali-treated gels corresponding to the alkali-untreated gels a,b. Lane CB shows the Coomassie blue-stained gel. Autoradiograms were exposed for 16 h.
Fig.2.
both
BIOCHEMICAL
3, 1991
of
samples (lanes of spectrin
under in
a ) and 7.3
these
the
conditions
two ghost
(lane
samples
b ) respectively. 1024
the
higher
in
the
than
that
in
spectrin
is
is much higher
when
effective
Mn2+
than
Ser-phosphorylation from
erythrocytes
of with
Vol.
178,
No.
BIOCHEMICAL
3, 1991
CB
Fig.3.
contrary,
in
higher
the
ghosts
that
in the
Fig.
3 shows
induced
in
that
the
the
pH is
internal
erythrocytes
a’
RESEARCH
b’
COMMUNICATIONS
c’
erythrocytes
the
increase
by
8 (lane re-adjusted
contrast,
the
by the
the
again
(lane
internal
pH was 7.3
internal
7.3
above pH changes
of
(lanes
pH
in
(lane
b') a').
their
completely
to 8 (lane
Ser-phosphorylation
slightly
Tyr-phosphorylation
internal is
is
less
pH was 8 (lane
3 protein b')
from
much
of Mn2+)
of band
to 7.3
3 (although
presence
whose
lowering
a')
of band
whose
from
ghosts
from
unaffected
c
Tyr-phosphorylation of Mg 2+ that in the
from
ghosts
erythrocytes By
the
presence
in the
than
b
a
BIOPHYSICAL
Autoradiograms showing the effect of lowering and subsequent increasing internal pH in human erythrocytes on the phosphoryJftion of membrane proteins in thei$+ghosts incubated with [y - P] ATP in the presence of 5 mM Mn . The ghosts were prepared from human erythrocytes whose internal pH was: 8 (Lanes a,a'); lowered from 8 to 7.3 (lanes b,b'); lowered to 7.3 and then re-adjusted to 8 (lanes c,c'). Lanes a',b', c' show the alkali-treated gels corresponding to the alkali-untreated gels a,b,c. Lane CB shows the Coomassie blue-stained gel. Autoradiograms were exposed for 16 h.
On the evident
AND
parent
reversed
when
c').
spectrin
appears
to
be
a,b,c,).
Conclusions The above erythrocytes the
results
may modify
phosphorylation When the
Tyr-kinase
This explain
internal
for
the the
that
internal
distribution
change
in
the
thus
human
regulating
proteins.
decreases
markedly
pH
of protein-kinases,
of membrane pH
is
whereas
responsible
practically
the
state
activity
counterpart, major
indicate
from
8
increased
to at
membrane-associated Ser-phosphorylation
7.3,
membrane-associated
expense
casein
of
kinase
of membrane
the
cytosolic
activity
proteins)
(the remains,
unchanged. different why changing
Ser-phosphorylation
membrane the
internal
of spectrin
binding
behaviour pH in human
and transmembrane 1025
of
the
above
erythrocytes band
enzymes from
3 in their
8 to
may 7.3
isolated
Vol.
BIOCHEMICAL
178, No. 3, 1991
ghosts in unaffected,
while the
AND BIOPHYSICAL
Tyr-phosphorylation
RESEARCH COMMUNICATIONS
of band 3 is
markedly
increased. Tyr-phosphorylation Previous studies (26) have shown that the in vitro of cytoplasmic domain of transmembrane band 3 protein of human erythrocytes enzymes to its N-terminal region. inhibits the binding of some glycolytic Such a binding of these enzymes, on the other hand, has been reported to lead to the inhibition
of their
Consequently the
activity
(27,28).
Tyr-phosphorylation of band 3, by inhibiting the enzymes, may result in an increase of glycolysis,
binding of someglycolytic
as suggested in (29). These results would suggest that
a variation
kinase to band 3 cytoplasmic domain, modifying be one reversible
mechanism by which a variation
of binding of
Tyr-protein
its Tyr-phosphorylation, of internal
might
pH may regulate
the glycolysis. Acknowledgments Miss Carla Munari for the technical assistance, Miss Anna Maria Monaco for the secretarial aid, Mr. Giorgio Parpajola for preparing the figures and Mr. Dario Spine110 for supplying
fresh blood from volunteers
are
gratefully
acknowledged. References 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Avruch J. and Fairbanks G. (1974) Biochemistry 13, 5507-5513. Hosey M.M. and Tao M. (1977) Biochim. Biophys. Acta 482, 348-357. Fairbanks G., Avruch J., Dino J.E. and Pate1 V.P. (1978) J. Supramol. Struct. 9, 97-112. Clari G., Michielin E. and Moret V. (1978) Biochim. Biophys. Acta 539, 420-426. Tao M., Conway R. and Cheta S. (1980) J. Biol. Chem. 255, 2563-2568. Simkowski K.W., and Tao M. (1980) J. Biol. Chem. 255, 6456-6461. Boivin P.and Galand C. (1980) Biochem. Biophys. Res. Commun.93, 24-28. Tao M., Conway R., Chiang H.C., Cheta S. and Yan T.F. (1981) in Rosen, O.M. and Krebs, E.G., eds.) Protein phosphorylation, Vol. 8 pp.1301-1312, Cold Spring Harbor Lab., NY. Conway R.G. and Tao M. (1981) J. Biol. Chem. 256, 11932-11938. Clari G. and Moret V. (1981) Biochim. Biophys. Acta 659, 370-377. Erusalimsky J.D., Balas N. and Milner Y. (1983) Biochim. Biophys. Acta 756, 171-181. Clari G. and Ferrari S. (1983) It. J. Biochem. 32. 174-188. Clari G. and Moret V. (1985) Mol. Cell. Biochem. 68, 181-187. Graham C., Avruch J. and Fairbanks G. (1976) Biochem. Biophys. Res. Commun.72, 701-708. Clari G. and Moret V. (1981) Biochem. Int. 2, 509-515. Usui H., Kinohara N., Yoshikawa K., Imazu M., Imaoka T. and Takeda M. (1983) J. Biol. Chem. 258, 10455-10463. Boivin P. and Galand C. (1986) Biochem. Biophys. Res. Commun. 134, 557-564. Clari G., Brunati A.M. and Moret V. (1986) Biochem. Biophys. Res. Commun.137, 566-572. Usui H., Imazu M., Maeta K., Tsukamoto H., Azuma K. and Takeda M. (1988) J. Biol. Chem. 263, 3752-3761. 1026
Vol.
20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Clari G., Marzaro G. and Moret V. (1990) Biochim. Biophys. Acta 1023, 319-324. Laemmli U.K. (1970) Nature (London) 227, 680-685. Bursaux E., Hilly M., Bluze A. and Poyart (1984) Biochim. Biophys. Acta 777, 253-260. Bradford M.M. (1976) Anal. Biochem. 72, 248-254. Clari G. and Moret V. (1989) Eur. J. Biochem. 179, 581-588. Clari G., Brunati A.M. and Moret V. (1988) Eur. J. Biochem. 175, 673-678. Low P.S., Allen D.P., Zioncheck T.F., Chari P., Willardson B.M., Geahlen R.L. and Harrison M.L. (1987) J. Biol. Chem. 262, 4592-4596. Murthy S.N.P., Liu T., Kaul R.K., Kohler H. and Steck T.L. (1981) J. Biol. Chem. 256, 11203-11208. Tsai J., Murthy S.N.P. and Steck T.L. (1982) J. Biol. Chem. 257, 1438-1442. Harrison M.L., Rathinavelu P., Arese P., Geahlen R.L. and Low P.S. (1991) J. Biol. Chem. 266, 4106-4111.
1027
178,
August
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1991
EFFECT OF
INTRACELLULAR
CHANGES ON THE DISTRIBUTION OF TYROSINE-
pH
SERINE/THREONINE-PROTEIN
G. CLARI,
Dipartimento
di
Biomembrane Received
June
KINASE ACTIVITIES
L. BORDIN,
Chimica
1021-1027
Biologica,
de1 Consiglio
AND
IN HUMAN ERYTHROCYTES
G. MARZARO and V. MORET
Universita Nazionale
di
delle
Padova
Ricerche,
and
Centro
Padova,
delle
Italy
1, 1991
SUMMARY: The pH-dependence of the distribution of Tyr-and Ser/Thr-protein kinases between cytosol and membrane in human erythrocytes was investigated. When the internal pH of human erythrocytes is decreased from 8 to 7.3 the membrane-associated Tyr-protein kinase activity markedly increases at expense of the cytosolic counterpart, whereas the membrane-bound and cytosolic casein kinase activity are unaffected. This different response of the two kinase activities to the imposed variation of intracellular pH may explain why the Tyr-phosphorylation of cytoplasmic domain of band 3 results to be much higher in the ghosts from erythrocytes whose internal pH was 7.3 than that in the ghosts from erythrocytes whose internal pH was 8. By contrast, the Ser-phosphorylation of spectrin o-subunit (band 2) and band 3 results to be practically unchanged in the ghosts from the erythrocytes treated at both pH values. 0 1991 Academic PTXL~S,Inc. Various
specific
corresponding distributed have
between
been
and other possible kinase
found
cytosol
of
The results
Tyr-protein
kinase
activity
counterpart
decreases
to
distribution two above presence is markedly
here
the
when
different of[y3-2
the
higher
the
in the
By contrast
the
erythrocytes
treated
8
same ghosts pH from
Ser-phosphorylation at both
the
distribution
values
from are
is
in
membrane the
cytosolic
intracellular
unaffected.
erythrocytes
whose
these
pH in the
the
separately
unchanged
of
a
membrane-associated while
of band
erythrocytes
of
intracellular the
pH
whether
state
practically
isolated
environment,
investigate
By contrast
remains
Tyr-phosphorylation ghosts
erythrocytes,
enhanced,
extent.
the
of human by ionic
when
as
differently
was to
to 7.3,
markedly
activity
intracellular P]ATP,
show that
as well
being
phosphorylation
from
is
of casein-kinase
Concomitantly,
the
decreased
is
study
may influence
regulating
reported
erythrocytes
in vitro
present pH
(l-13)
besides
structures
affected
intracellular thus
kinases
(14-19),
and membrane
The aim of the
activities,
intact
Tyr-protein
phosphatases
to be differently
factors. change
proteins.
Ser/Thr-and
specific
having
incubated 3 cytoplasmic internal the
the
in
the
domain pH was 7.3.
ghosts
from
the
pH values. 0006-291X/91
1021
$1.50
Copyrighr 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
Methods
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Materials
Human erythrocytes were prepared by centrifugation (at 750 x g for 3 min) of fresh blood collected from healthy donors. To minimize contamination by leucocytes and platelets, the packed red cells were washed three times by centrifugation in buffer A (50 mM Hepes pH 8, 1.5 mM MgSO , 90 mM KCl, 48.5 mM Na2S04, 0.1 mM EGTA, 25 pg/ml chloramphenicol, 0.1 mg/m f streptomycin, 12 mM Glucose and 1 mM Adenosine) and discarding the buffy coat and the upper third of packed red cell layer. Packed red cells were divided into 3 samples at a 10% hematocrit: i.e. in the sample 1, 1 ml packed red cells was suspended in 9 ml buffer A pH 8, while in the samples 2 and 3 3 1 ml packed red cells was suspended in 9 ml for 30 min buffer A adjusted to pH 6.9. The three samples were then incubated 1 and 7.3 for at O°C so that the controlled internal pH were 8 for sample both samples 2 and 3. Subsequently the packed cells recovered by centrifugation of samples 1 and 2 were hemolysed in 28 ml hypotonic 5 mM phosphate buffer (pH 8 for sample 1 and respectively pH 7.3 for sample 2) containing 0.02% NaN and 0.03 (PMSF). On the contrary the pat 9 ed cells mM phenylmethylsulphonyl fluoride were resuspended and incubated for further recovered from sample 3 (pH 7.3) 30 min at O°C in 9 ml buffer A adjusted to pH 8.5, in order to obtain again the internal pH 8. These latter cells, recovered by centrifugation, were then hemolysed in 28 ml of the above hypotonic buffer pH 8. separately recovered from hemolysate of The membranes and cytosol were the three above samples by centrifugation (at 20.000 x g for 20 min). The membrane were then washed twice in the same hypotonic lysis buffer containing 30 p Vanadate (pH 8 for samples 1 and 3 and pH 7.3 for sample 2) and twice in 25 mM TRIS-HCl buffer (pH 8 for the sample 1 and 3 and pH 7.3 for sample 2) containing 0.03 mM PMSF, 0.02% NaN3 and 30 pM Vanadate. Endogqqous phoshorylation of membrane proteins in the ghosts in the presence of [y"-'P'] ATP Endogenous Ser-phosphorylation of membrane proteins was tested by incubating the white ghosts (80 ug proteins) at 30 C for 5 min in 125 ul fXa~i~,~ix~~Ze,so~~~? JO0 mM Hepeg-HCl buffer (pH 7.5), l~yk!p~~;~a;;l P] ATP (6.10 cpm/nmol). Endogenous lation of gembrane proteins was assayed under the same conditions followed for Ser-phosphorylation, except that MgC12 was replaced by 5 mM MnCl . Incubation was stopped by addition of 2% SDS and 1% @-mercap z oethanol (final concentration) followed by a 5 min treatment at 100°C as described in (20). 40 g of solubilized membranes were analyzed by 0.1% SDS -10% PAGE essential P y according to (21). as previously described (20). Membrane-associated and cytosolic casein-kinase activities were assayed by incubating 80 pg of white ghosts and, respectively, 50 ul of dialyzed hemolysate supernatant prepared as described in (20), in the presence of 0.65 mg/ml whole casein as substrate, under the conditions above described for the endogenous Ser-phosphorylation of membrane proteins in the isolated ghosts. After 5 min incubation, the reaction was stopped as above described. Half of reaction mixture was analyzed by electrophoresis followed by autoradiography ag2described in (20). P-labelled casein band, identified by autoradiography, was excised from the gels and counted for radioactivity in a liquid scintillation counter. Membrane-bound and cytosolic Tyr-protein kinase activities were assayed by incubating at 30°C for 5 min, 80 pg of white ghosts and, respectively, 50 1 of dialyzed ammonium-sulfate fraction (60% saturation) prepared from rl emolysate supernatant as described in (20), in the presence of 8 pg poly Tyr) 4:1, as substrate, under the (Glu, same conditions followed for the endogenous Tyr-phosphorylation of membrane proteins. The incubation was stopped and half of reaction mixture was analyzed by electrophoresis followed by autoradiography, as previously degsribed in (20). The corresponding diffuse P-Tyr-labelled band, identified by autoradiography, was excised from the alkali-treated gels and counted for radioactivity in a liquid scintillation counter.
1022
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Other
methods; Miscellaneous Internal pH was assayed according to Borseaux (22). Protein content was determined according to Bradford (23). Synthetic polymer poly (Glu, Tyr) 4:l (molecu 2r mass ranging from 20 kDa to 50 kDa) was purchased from Sigma; [ 4 -PI ATP from Amersham International (U.K.) and whole casein was prepared from commercial pow&r (Merck) as previously described (4). Results When the 7.3
internal
a different
Tyr-protein
kinase
Tyr-kinase
activity,
increased,
while
(See
I).
Table whole
the
above
the
two
explain
the
1) observed
distribution
ghosts
internal
pH was 8 and 7.3
presence
of 5 mM Mn2+.
Tyr-and
are
incorporated
poly
(Glu,Tyr)
Membrane
Human with
is
as into
activity
practically a different of
the
incubated
same
extent
(assayed
on by
response
of
pH
patterns
may (Fig.
samples
whose in
the cytosolic and in human erythrocytes
Ser-protein kinase activity (expressed cpm incorporated
4:l)
whole
Cytosol
Membrane
as into
casein) Cytosol
erythrocytes internal
pH 8
1500
3925
9000
36000
2650
2750
8900
35500
Human erythrocytes with
internal
pH 7.3
The Tyr and Ser protein kinase activities were assayed (as described in in the ghosts prepared from 10 ~1 Methods and Materials) in the cytosol and of two samples of packed human erythrocytes having internal pH 8 and pH 7.3 as countslmin incorporated respectively.3Tyr-kinase activity was expressed P-labelled electrophoretic band of Poly (Glu,Tyr) 4~1 under into diffuse conditions described in Methods and Materials. The Ser protein kinase into whole casein under activity is expressed as counts/min incorporated conditions described in Methods and Materials.
1023
be
unaffected
with[@PlATP
on
pH of
to
intracellular
erythrocyte
pH change kinase activities
Tyr-protein kinase activity (expressed
pH 8 to
results
to the
Ser-phosphorylation
respectively,
Effect of intracellular I. membrane-bound Tyr-and Ser-protein
4:1,
kinase
variation
from
TABLE
from
decreased
of pH. Such
isolated
cpm
Tyr)
and cytosol to the
endogenous the
(Glu, is
change
activities
different
poly
lowered
intracellular distribution i.e. the membrane-associated
of casein
membrane
intracellular
kinase when
the
counterpart
between
mentioned
on
cytosolic
the
is
exhibited by the kinase activities:
assayed the
casein)
protein
human erythrocytes
is
and casein
By contrast, added
pH of
response
the
Vol.
178,
No.
tea
01 Pig.1.
The
a'
b
32
P-labelling
erythrocyte
of
Tyrosyl-residues
is
32P-labelled 1
from
ghosts By
in the is
a
clearly
a'
b
b’
from
its
of
alkali-treated
gels, due
partial
of
2) in
the
to
that
the
spectrin
remains
internal
pH 8 (lane
from
while
the
as
phosphorylation and
of confirmed
alkali-treated
internal
whose
is
pH was 7.3
internal
phosphorylation
of
also
unchanged
much
(lane
b'),
pH was 8 (lane
a').
Seryl-residues
a,b). and band 3 the ghosts are incubated in presence of 10 mM Mg 2+ (more in activating the endogenous Ser-casein-kinase). that
ghosts residues
alkali-stability
Tyr-phosphorylation
whose
erythrocytes
2 shows
the seryl
(24,25).
shows
erythrocytes
contrast
(band
phosphorylation
partially
by
practically unchanged in both Such a Ser-phosphorylation
Fig.
COMMUNICATIONS
RESEARCH
CB
@-subunit
analysis
membranes
Fig. the
BIOPHYSICAL
02
the
due to
as indicated
32 P-phosphoaminoacid
ghosts
b’
spectrin
samples
indicated by its disappearance 32 P-labelling of band 3 by
AND
Autoradiograms showing the effect of variation of internal pH of human erythrocytes on the phosphoryl.at\fn of membrane proteins in the ghof$s incubated 5 min. with [r-P] ATP in the presence of5mMMn . The ghosts were prepared from the two samples of human erythrocytes having internal pH 8 (lanes a,a') and 7.3 (lanes b,b') respectively. Lanes a',b' show the alkali-treated gels corresponding to the alkali-untreated gels a,b. Lane CB shows the Coomassie blue-stained gel. Autoradiograms were exposed for 16h. Autoradiograms showing the effect of variation of internal pH in human erythrocytes on the phosphorylation of mpbrane proteins in their ghost?+ incubated for 5 min. with [Y- P] ATP in the presence of Mg (10 mM). The ghosts were prepared from the two samples of human erythrocytes having internal pH 8 (lanes a,a') and 7.3 (lanes b,b'), respectively. Lanes a',b' show the alkali-treated gels corresponding to the alkali-untreated gels a,b. Lane CB shows the Coomassie blue-stained gel. Autoradiograms were exposed for 16 h.
Fig.2.
both
BIOCHEMICAL
3, 1991
of
samples (lanes of spectrin
under in
a ) and 7.3
these
the
conditions
two ghost
(lane
samples
b ) respectively. 1024
the
higher
in
the
than
that
in
spectrin
is
is much higher
when
effective
Mn2+
than
Ser-phosphorylation from
erythrocytes
of with
Vol.
178,
No.
BIOCHEMICAL
3, 1991
CB
Fig.3.
contrary,
in
higher
the
ghosts
that
in the
Fig.
3 shows
induced
in
that
the
the
pH is
internal
erythrocytes
a’
RESEARCH
b’
COMMUNICATIONS
c’
erythrocytes
the
increase
by
8 (lane re-adjusted
contrast,
the
by the
the
again
(lane
internal
pH was 7.3
internal
7.3
above pH changes
of
(lanes
pH
in
(lane
b') a').
their
completely
to 8 (lane
Ser-phosphorylation
slightly
Tyr-phosphorylation
internal is
is
less
pH was 8 (lane
3 protein b')
from
much
of Mn2+)
of band
to 7.3
3 (although
presence
whose
lowering
a')
of band
whose
from
ghosts
from
unaffected
c
Tyr-phosphorylation of Mg 2+ that in the
from
ghosts
erythrocytes By
the
presence
in the
than
b
a
BIOPHYSICAL
Autoradiograms showing the effect of lowering and subsequent increasing internal pH in human erythrocytes on the phosphoryJftion of membrane proteins in thei$+ghosts incubated with [y - P] ATP in the presence of 5 mM Mn . The ghosts were prepared from human erythrocytes whose internal pH was: 8 (Lanes a,a'); lowered from 8 to 7.3 (lanes b,b'); lowered to 7.3 and then re-adjusted to 8 (lanes c,c'). Lanes a',b', c' show the alkali-treated gels corresponding to the alkali-untreated gels a,b,c. Lane CB shows the Coomassie blue-stained gel. Autoradiograms were exposed for 16 h.
On the evident
AND
parent
reversed
when
c').
spectrin
appears
to
be
a,b,c,).
Conclusions The above erythrocytes the
results
may modify
phosphorylation When the
Tyr-kinase
This explain
internal
for
the the
that
internal
distribution
change
in
the
thus
human
regulating
proteins.
decreases
markedly
pH
of protein-kinases,
of membrane pH
is
whereas
responsible
practically
the
state
activity
counterpart, major
indicate
from
8
increased
to at
membrane-associated Ser-phosphorylation
7.3,
membrane-associated
expense
casein
of
kinase
of membrane
the
cytosolic
activity
proteins)
(the remains,
unchanged. different why changing
Ser-phosphorylation
membrane the
internal
of spectrin
binding
behaviour pH in human
and transmembrane 1025
of
the
above
erythrocytes band
enzymes from
3 in their
8 to
may 7.3
isolated
Vol.
BIOCHEMICAL
178, No. 3, 1991
ghosts in unaffected,
while the
AND BIOPHYSICAL
Tyr-phosphorylation
RESEARCH COMMUNICATIONS
of band 3 is
markedly
increased. Tyr-phosphorylation Previous studies (26) have shown that the in vitro of cytoplasmic domain of transmembrane band 3 protein of human erythrocytes enzymes to its N-terminal region. inhibits the binding of some glycolytic Such a binding of these enzymes, on the other hand, has been reported to lead to the inhibition
of their
Consequently the
activity
(27,28).
Tyr-phosphorylation of band 3, by inhibiting the enzymes, may result in an increase of glycolysis,
binding of someglycolytic
as suggested in (29). These results would suggest that
a variation
kinase to band 3 cytoplasmic domain, modifying be one reversible
mechanism by which a variation
of binding of
Tyr-protein
its Tyr-phosphorylation, of internal
might
pH may regulate
the glycolysis. Acknowledgments Miss Carla Munari for the technical assistance, Miss Anna Maria Monaco for the secretarial aid, Mr. Giorgio Parpajola for preparing the figures and Mr. Dario Spine110 for supplying
fresh blood from volunteers
are
gratefully
acknowledged. References 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Avruch J. and Fairbanks G. (1974) Biochemistry 13, 5507-5513. Hosey M.M. and Tao M. (1977) Biochim. Biophys. Acta 482, 348-357. Fairbanks G., Avruch J., Dino J.E. and Pate1 V.P. (1978) J. Supramol. Struct. 9, 97-112. Clari G., Michielin E. and Moret V. (1978) Biochim. Biophys. Acta 539, 420-426. Tao M., Conway R. and Cheta S. (1980) J. Biol. Chem. 255, 2563-2568. Simkowski K.W., and Tao M. (1980) J. Biol. Chem. 255, 6456-6461. Boivin P.and Galand C. (1980) Biochem. Biophys. Res. Commun.93, 24-28. Tao M., Conway R., Chiang H.C., Cheta S. and Yan T.F. (1981) in Rosen, O.M. and Krebs, E.G., eds.) Protein phosphorylation, Vol. 8 pp.1301-1312, Cold Spring Harbor Lab., NY. Conway R.G. and Tao M. (1981) J. Biol. Chem. 256, 11932-11938. Clari G. and Moret V. (1981) Biochim. Biophys. Acta 659, 370-377. Erusalimsky J.D., Balas N. and Milner Y. (1983) Biochim. Biophys. Acta 756, 171-181. Clari G. and Ferrari S. (1983) It. J. Biochem. 32. 174-188. Clari G. and Moret V. (1985) Mol. Cell. Biochem. 68, 181-187. Graham C., Avruch J. and Fairbanks G. (1976) Biochem. Biophys. Res. Commun.72, 701-708. Clari G. and Moret V. (1981) Biochem. Int. 2, 509-515. Usui H., Kinohara N., Yoshikawa K., Imazu M., Imaoka T. and Takeda M. (1983) J. Biol. Chem. 258, 10455-10463. Boivin P. and Galand C. (1986) Biochem. Biophys. Res. Commun. 134, 557-564. Clari G., Brunati A.M. and Moret V. (1986) Biochem. Biophys. Res. Commun.137, 566-572. Usui H., Imazu M., Maeta K., Tsukamoto H., Azuma K. and Takeda M. (1988) J. Biol. Chem. 263, 3752-3761. 1026
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178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Clari G., Marzaro G. and Moret V. (1990) Biochim. Biophys. Acta 1023, 319-324. Laemmli U.K. (1970) Nature (London) 227, 680-685. Bursaux E., Hilly M., Bluze A. and Poyart (1984) Biochim. Biophys. Acta 777, 253-260. Bradford M.M. (1976) Anal. Biochem. 72, 248-254. Clari G. and Moret V. (1989) Eur. J. Biochem. 179, 581-588. Clari G., Brunati A.M. and Moret V. (1988) Eur. J. Biochem. 175, 673-678. Low P.S., Allen D.P., Zioncheck T.F., Chari P., Willardson B.M., Geahlen R.L. and Harrison M.L. (1987) J. Biol. Chem. 262, 4592-4596. Murthy S.N.P., Liu T., Kaul R.K., Kohler H. and Steck T.L. (1981) J. Biol. Chem. 256, 11203-11208. Tsai J., Murthy S.N.P. and Steck T.L. (1982) J. Biol. Chem. 257, 1438-1442. Harrison M.L., Rathinavelu P., Arese P., Geahlen R.L. and Low P.S. (1991) J. Biol. Chem. 266, 4106-4111.
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