Vol. 84, No. 3, 1978 October
BIOCHEMICAL
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
16, 1978
Pages
660-666
SHORT-TIMEPHOSPHORYMTION OF PLASMAMEMBRANE PROTEINSBY ENXXXNOUSKINASES Thorns Joos and F.AlfredAnderer Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, 7400 Tiibingen, Fed.Rep.Germany Received
August
18,
1978
Sumnary: Endogenousprotein phosphorylation SV 40-transfo&xed muse fibroblasts
in plasma membranesisolated from
was studied in the presence and absence
of cyclic nucleotides. Using low concentrations of membraneprotein the kinetics of ATP-dependent 32P-incorporation showeda rapid phosphorylation reaction up to 2 set of incubation which was stmated by cAMP and inhibited by cGMP. This short-time phosphorylation reaction was followed by a rapid dephosphorylation and a slower rephosphorylation. ent on protein concentration. The perspectives on cellular
This phenomenonwas depend-
phosphoproteins have changed dramatically
with
the discovery of CAMP- and cGMP-dependent protein kinases (1,2).
A general
hypothesis is that protein phosphorylation
represents
a potential
mechanismfor the control
isms which might be mediated by cyclic mation on the velocity tigations
of membraneproteins (3-12),
of membraneevents in eukaryotic nucleotides.
of such regulatory
on the effects
of cyclic
and dephosphorylation
Only very little
reactions
is available.
information
inforMost inves-
nucleotides on endogenous phosphorylation
show linear kinetics
up to an incubation time of 5-10 min
someup to 2 min (13-16). However, these kinetic
detailed
organ-
on phosphorylation/dephosphorylation
data do not give reactions
in the
tin-e range of a few seconds. Since transmission or modulation of regulatory short-time
signals might be of special biological
short-time kinetics
of endogenousprotein
importance we studied the
phosphorylation
ma membranesof SV 40-transformed mousefibroblasts sence of cyclic
in the presence and ab-
(lo-40
ANDMEX'HODS
Ci/mml),[~-3*P]GTP
OOOS-291X/78/0843~0660$01.00/0 Copyright All rights
plas-
nucleotides. I%!ENNS
Cbnicals:[\r-a*P]ATP
with purified
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
660
(lo-40
Ci/ml)
were
purchased
Vol. 84, No. 3, 1978
BIOCHEMICAL
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
from NEN, Boston, USA. Aminophylline [(theophylline)2.ethylenediamine] and ethyleneglycol-bis-(f&aminoethyl ether)N'N-tetracetic acid (EGTA) were purchased from Si@a, St.Louis,
USA; adenosine 3':5'-cyclic
monophosphate(CAMP)
and guanosine 3':5'-cyclic monophosphate(cGMP) from Boehringer, Mannheim, Germany. NCStissue solubilizer was obtained from Amersham/Searle, Arlington, Ill.,
USA. All other chemicals were of analytical
Germany). Kallilaein
inactivator
(17) was a gift
grade (Merck, Baxmstadt, from Bayer, Elberfeld,
Ger-
WY* Cells and Preparation of MembraneFractions: All investigations were performed with STU-5lA/232B mousefibroblasts, a subline derived from SV 40transformed embryonic cells of STUmice. The cells were grown in suspension culture, harvested in the exponential growth phase and washed 3 times with saline and once with 1mMMgCl . Thereafter, the cells were resuspended in lmM MgC12for 5 min and 5x1072cells/ml were disrupted in a Dxnce homogenizer. Nuclei and still intact cells were removed by centrifugation at 2OOxgand 4OC for 2 min. Thereafter, all membranousmaterial was sedimented to a pellet at 30 000 rpm and 4OCfor 30 min using a Spinco rotor Ti 30. The pellet was suspended in 5% sucrose, layered onto a continous gradient of 20-60% sucrose and centrifuged in a Spinco SW40 rotor at 30 000 rpm and 4OCfor 150 min. The membranematerial of the upper band at about 30% sucrose, identified as plasma membranefraction by marker enzyme assays, was sedimented to a pellet in a Spinco rotor Ti 30 at 30 000 qxn and 4OCfor 60 min.The sucrose gradient centrifugation
was repeated and the resulting
plasma membranefraction
showeda 20-fold increase of the specific activity of phosphodiesterase and alkaline phosphatase. The final sediment was washed once with water and used for phosphorylation experiments without delay. Phosphorylation of MembraneFractions: Phosphorylation was carried out at 37OCin a total volume of 9Opl using a glycine/phosphate buffer pH 7.8 (adjusted at 37OC) which contained EGTAand cyclic nucleotides. The individual incubation mixtures were prepared by mixing 10 ~1 of buffer solution, lo ~1 of MgC12 solution, 20 ~1 of membranesuspension and 50 ~1 containing 50 uCi of[y-32P]ATP
or [f -s2PJGTP, respectively.
The final
concentrations
were:
25 mMglycine, 25 mMphosphate, 0.3 mMEGTA, 10 mMMgCl,, CAMPor cGMPof the given concentration between 10°4M and lo-'M and 1 n-Maminophylline. In addition each sample contained 9ug of kallikrein inactivator to block endogenous pro-teases. The final and 240 ug.
content of membraneproteins ranged between 24
All solutions were mixed and preincubated at 37OCfor 1 min except the solutions of the labeled trinucleotides which were added thereafter thus marking
661
Vol. 84, No. 3, 1978
8lOCHEMlCAL
zerotime of the phosphorylation
AND BIOPHYSICAL
reaction.
RESEARCH COMMUNICATIONS
The endpoint of the reaction
was
marked by addition of 10 ~1 of 0.1 M phosphate buffer/O.1 M EDTA pH 7.2 conptoethanol, followed at once by boiling at taining 10% SDS and 10%13-merca 100°C for 5 min. Thereafter
the samples were brought to 10%of glycerol
and
used directly for SDSpolyacrylamide gel electrophoresis. SDS-gel electrophoresis was performed in 5% polyacrylamide gels (5x90 nun>. Gels were cut into 1.5 mmslices and extracted
with NCS solubilizer.
RESULTS Effects
of concentration
of cyclic
Endogenousphosphorylation
nucleotides on protein phospl-mylation:
of the plasm membranefraction
with [~-~~P]ATP
was allowed to proceed for 1 min at pH 7.8 and 37OCin the presence of cyclic AMP or cyclic overall
&P, 10 mMMgC12and lti
phosphorylation
exhibited
AMP (Fig.11 and 10m5Mcyclic phylline
concentration
aminophylline.
Under these conditions
maxima at concentrations
WP, respectively.
to 10 r&l resulted
of 10V7Mcyclic
An increase of the amino-
in a 40-50% reduction
of mernbrane-
bound [32P]phosphate at the conditions of maximm incorporation. Experimnts
performed with [r -3*P]GTP showedthat the GTP-dependent overall
phosphorylation addition
of the plam
of cyclic
Short-time kinetics
membranewas not significantly
W or cyclic
influenced by
AMP.
and the effect
of protein concentration:
The kinetic
periments were performed under standard conditions using concentrations cyclic
nucleotides which had shown optiml
lation
after
1 min incubation.
tein-bound
of overall
phosphory-
described was 2+0.5 sec. Further
were 5, 15, 60 and 600 sec. In Fig.2 the data of pro-
p*P]phosphate are given for a low concentration
teins (24ug per sample). The time courses of phosphorylation or absence of cyclic
of
The shortest incubation time which could be
achieved under the experimental conditions incubation intervals
stimlation
ex-
nucleotides
indicate
of membraneproin the presence
a rapid phosphorylation
followed by a rapid dephosphorylation and a slow rephosphorylation started between 5 and 15 sec. The stirmlation in the presence of cyclic
AMP after
of short-tim
up to 2 set which
phosphorylation
2 set incubation ranged between 70-100'10
662
Vol. 84, No. 3, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH CO/vVvtUNlCATlONS
cl
i
0
1
b Cyclic 5
1. Effects
6 nucleotide
'
of cyclic
I,
25
02
C-logB Ml *
Eal
M) Incubation
nucleotide concentration
plasma membraneproteins. under standard conditions
L
15
time
Csecl
on [32P]incorporation
into
Phosphorylation reactions were carried out using [y -J"P]ATP as radioactive precursor.
Incubation time was 1 min, the content of membraneprotein was 108 pg/ sample. Effect
of cAMP (
l
1; effect
of cGMP( o ).
2. Effect of incubation time on phosphorylation/dephosphorylation kinetics in the absence and presence of cyclic nucleotides using a low plasma membraneprotein concentration (24 ug/sample) and [T-~~P~ATP as precursor. ( l 1 without cyclic nucleotides; (V') 10-7M CAMP; (1) 10-5M cGMP.
while cyclic
GMF'showeda 20-30% inhibition
under these conditions.
5 set incubation it appeared that the presence of cyclic phosphorylation
yielding
compared to the control rephosphorylation,
After
GMPstimulated de-
only lo-40% of protein bound [3*P]phosphate when or to incubation with cyclic
after
5 to 15 set incubation,
nucleotides did not have a very specific
effect
AMP. In the phase of
the presence of cyclic on the time course of phos-
phorylation. The pattern of phosphoproteins obtained after phoresis was not significantly seen in Fig.3,
the relative
incubation is very similar
SDSpoly-acrylamide
influenced by cyclic distribution
nucleotides.
of phosphoproteins after
in the presence and absence of cyclic
663
gel electroAs can be 2 set nucleotides
BIOCHEMICAL
Vol. 84, No. 3, 1978
166
110
B
U
Z?
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
16
6
Ol 3
’
Ekctroph&c6mipmtion
hil
3. SDSpolyacrylmide
I 60
15
0
-I
Incubation
gel (5%) electrophoresis
time
of the plasm membrane
fraction, after 2 set of incubation of a low concentration membraneproteins (42pg/sample) under standard conditions. Without cyclic 4. Effect
nucleotides;
600
Csecl
( o ) 10-7M CAMP; (
l
of plasma Cotted line:
> lo-'M &ML'.
of incubation time on phosphorylation/dephosphorylation
kinetics
in the absence and presence of cyclic nucleotides using a high plasm m&x?ane protein concentration (240ug/sample) and[y-32P]ATP as precursor. (
> without cyclic
l
and differs
essentially
porated. The pattern
nucleotides;
also resembled that obtained after
by cyclic
60 set mximum stimlation of cyclic
were obtained
of membraneproteins was increased by a factor phosphorylation
AMP as well as by cyclic
10
and dephosphory-
could be no more recognized but late-phase phosphorylation
found to be stimlated
all
1 min incubation.
tima courses of phosphorylation
(24Opg per sample). The phase of short-time lation
10m5M&HP.
only in the absolute amount of [32Plphosphate incor-
Somewhatdifferent when the concentration
(V > 10W7MCAMP; (I)
was now
G&IF'(Fig.4).
After
was observed ranging between 20-708 in the presence
AMP and between IO-50% in the presence of cyclic
tin-e courses show a phase where dephosphorylation
mre rapid in the presence of cyclic
W.
predominatly occurred,
nucleotides than without.
664
Thereafter,
BIOCHEMICAL
Vol. 84, No. 3, 1978
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION The phenomenonof short-time
phosphorylation/dephosphorylation
brane proteins might represent a biologically The phosphorylation concentrations lation.
data obtained after
do not necessarily
significant
phosphorylation
is possibly achieved
slow rephosphorylation
with the linear phosphorylation kinetics
Short time phosphorylation
of membraneproteins,
phosphoprotein patterns no specific
phosphorylation
is difficult
described by a
stimulated by cyclic
nucleo-
As far as it can be judged from the
preference of one or the other membrane
The influence of high concentrations of phosphorylation
corre-
(3-16).
tides or not, appears to be not selective.
protein during short-time
phosphory-
already part of the rapid dephosphorylation
in less than one second. The phase of relatively
number of other investigators
system.
2 set incubation with low protein
time course and the maximumof short-tin-e
indicate
regulatory
correspond to maximumshort-time
These data are most likely
lates most likely
of plasma mem-
is evident.
of membraneproteins on the time course
to interpret.
Our data do not necessarily
that short-time phosphorylation/dephosphorylation
might not have
occurred in this case since the phase of rapid dephosphorylation could have already come to the end after phosphorylation
2 set incubation.
of high concentrations
stimulated in the presence of cyclic tions late-phase stinuiLation by cyclic only after
10 min incubation.
On the other hand, late-phase
of membraneproteins was found to be nucleotides.
At low protein concentra-
nucleotides attained
values
A possible explanation of this phenomenonis
offered by the assumption that increasing concentrations are speeding up the time course of short-time lation
significant
of membraneprotein
phosphorylation/dephosphory-
and rephosphorylation. REFERENCES
and E.G.Krebs (1968) J.Biol.Chem. 243, 3763-3765. 2. Kuo, J.F. and P.Greengard (1970) J.Biol.Chem. 245, 2493-2498. 3. Iabrie, F., S.Lemaire, G.Poirier, G.Pelletier and R.Boucher (1971) J.Biol. them. 246, 7311-7317. 1. Walsh, D.A., J.P.Perkins
665
vol.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
84, NO. 3, i 978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Guthrow,C.E., J.E.Allen and H.Rasmussen (1972) J.Biol.Chem. 247, 8145-8153. Rubin,C.S. and O.M.Rosen (1973) Bicchem.Biophys.Res.km. 50, 421-429. Bacalao, J. and M.Rieber (1973) Febs Letters 37, 37-41. Azhar, S. and K.M.J.Menon (1975) Bicchem.J. 151, 23-26. Steiner, M. (1975) Psch.Biochem.Biophys. 171, 245-254. Hosey, M.M. and M.Tao (1976) Biochemistry 5, 1561-1568. Mast-ro, A.M. and E.Rozengurt (1976) J.Biol.Chem. 251, 7899-7906. George, E.R., R.A.BalaJcir, C.R.Filburn and B.Saclctor (1977) Arch.Biochem. Biophys. 180, 429-443. Branton, P.E. and J.Laudry-Magnan (1978) Biochim.Biophys.Acta 508, 246-259. Miyax&o,E. and S.Kakiuchi (1974) J.Biol.Chem. 249, 2769-2777. Avruch,J. and G.Fai.rbanks (1974) Biochemistry 3, 5507-5514. Chang, K.J., N.A.Marcus and P.Cuatrecacas (1974) J.Biol.Chem. 249, 68546865. Singh, H. and N.Spritz (1976) Biochim.Biophys.Acta 3, 325-337. Anderer, F.A. and S.H&nle (1966) J.Biol.Chem. 241, 1568-1572.
666
BIOCHEMICAL
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
16, 1978
Pages
660-666
SHORT-TIMEPHOSPHORYMTION OF PLASMAMEMBRANE PROTEINSBY ENXXXNOUSKINASES Thorns Joos and F.AlfredAnderer Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, 7400 Tiibingen, Fed.Rep.Germany Received
August
18,
1978
Sumnary: Endogenousprotein phosphorylation SV 40-transfo&xed muse fibroblasts
in plasma membranesisolated from
was studied in the presence and absence
of cyclic nucleotides. Using low concentrations of membraneprotein the kinetics of ATP-dependent 32P-incorporation showeda rapid phosphorylation reaction up to 2 set of incubation which was stmated by cAMP and inhibited by cGMP. This short-time phosphorylation reaction was followed by a rapid dephosphorylation and a slower rephosphorylation. ent on protein concentration. The perspectives on cellular
This phenomenonwas depend-
phosphoproteins have changed dramatically
with
the discovery of CAMP- and cGMP-dependent protein kinases (1,2).
A general
hypothesis is that protein phosphorylation
represents
a potential
mechanismfor the control
isms which might be mediated by cyclic mation on the velocity tigations
of membraneproteins (3-12),
of membraneevents in eukaryotic nucleotides.
of such regulatory
on the effects
of cyclic
and dephosphorylation
Only very little
reactions
is available.
information
inforMost inves-
nucleotides on endogenous phosphorylation
show linear kinetics
up to an incubation time of 5-10 min
someup to 2 min (13-16). However, these kinetic
detailed
organ-
on phosphorylation/dephosphorylation
data do not give reactions
in the
tin-e range of a few seconds. Since transmission or modulation of regulatory short-time
signals might be of special biological
short-time kinetics
of endogenousprotein
importance we studied the
phosphorylation
ma membranesof SV 40-transformed mousefibroblasts sence of cyclic
in the presence and ab-
(lo-40
ANDMEX'HODS
Ci/mml),[~-3*P]GTP
OOOS-291X/78/0843~0660$01.00/0 Copyright All rights
plas-
nucleotides. I%!ENNS
Cbnicals:[\r-a*P]ATP
with purified
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
660
(lo-40
Ci/ml)
were
purchased
Vol. 84, No. 3, 1978
BIOCHEMICAL
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
from NEN, Boston, USA. Aminophylline [(theophylline)2.ethylenediamine] and ethyleneglycol-bis-(f&aminoethyl ether)N'N-tetracetic acid (EGTA) were purchased from Si@a, St.Louis,
USA; adenosine 3':5'-cyclic
monophosphate(CAMP)
and guanosine 3':5'-cyclic monophosphate(cGMP) from Boehringer, Mannheim, Germany. NCStissue solubilizer was obtained from Amersham/Searle, Arlington, Ill.,
USA. All other chemicals were of analytical
Germany). Kallilaein
inactivator
(17) was a gift
grade (Merck, Baxmstadt, from Bayer, Elberfeld,
Ger-
WY* Cells and Preparation of MembraneFractions: All investigations were performed with STU-5lA/232B mousefibroblasts, a subline derived from SV 40transformed embryonic cells of STUmice. The cells were grown in suspension culture, harvested in the exponential growth phase and washed 3 times with saline and once with 1mMMgCl . Thereafter, the cells were resuspended in lmM MgC12for 5 min and 5x1072cells/ml were disrupted in a Dxnce homogenizer. Nuclei and still intact cells were removed by centrifugation at 2OOxgand 4OC for 2 min. Thereafter, all membranousmaterial was sedimented to a pellet at 30 000 rpm and 4OCfor 30 min using a Spinco rotor Ti 30. The pellet was suspended in 5% sucrose, layered onto a continous gradient of 20-60% sucrose and centrifuged in a Spinco SW40 rotor at 30 000 rpm and 4OCfor 150 min. The membranematerial of the upper band at about 30% sucrose, identified as plasma membranefraction by marker enzyme assays, was sedimented to a pellet in a Spinco rotor Ti 30 at 30 000 qxn and 4OCfor 60 min.The sucrose gradient centrifugation
was repeated and the resulting
plasma membranefraction
showeda 20-fold increase of the specific activity of phosphodiesterase and alkaline phosphatase. The final sediment was washed once with water and used for phosphorylation experiments without delay. Phosphorylation of MembraneFractions: Phosphorylation was carried out at 37OCin a total volume of 9Opl using a glycine/phosphate buffer pH 7.8 (adjusted at 37OC) which contained EGTAand cyclic nucleotides. The individual incubation mixtures were prepared by mixing 10 ~1 of buffer solution, lo ~1 of MgC12 solution, 20 ~1 of membranesuspension and 50 ~1 containing 50 uCi of[y-32P]ATP
or [f -s2PJGTP, respectively.
The final
concentrations
were:
25 mMglycine, 25 mMphosphate, 0.3 mMEGTA, 10 mMMgCl,, CAMPor cGMPof the given concentration between 10°4M and lo-'M and 1 n-Maminophylline. In addition each sample contained 9ug of kallikrein inactivator to block endogenous pro-teases. The final and 240 ug.
content of membraneproteins ranged between 24
All solutions were mixed and preincubated at 37OCfor 1 min except the solutions of the labeled trinucleotides which were added thereafter thus marking
661
Vol. 84, No. 3, 1978
8lOCHEMlCAL
zerotime of the phosphorylation
AND BIOPHYSICAL
reaction.
RESEARCH COMMUNICATIONS
The endpoint of the reaction
was
marked by addition of 10 ~1 of 0.1 M phosphate buffer/O.1 M EDTA pH 7.2 conptoethanol, followed at once by boiling at taining 10% SDS and 10%13-merca 100°C for 5 min. Thereafter
the samples were brought to 10%of glycerol
and
used directly for SDSpolyacrylamide gel electrophoresis. SDS-gel electrophoresis was performed in 5% polyacrylamide gels (5x90 nun>. Gels were cut into 1.5 mmslices and extracted
with NCS solubilizer.
RESULTS Effects
of concentration
of cyclic
Endogenousphosphorylation
nucleotides on protein phospl-mylation:
of the plasm membranefraction
with [~-~~P]ATP
was allowed to proceed for 1 min at pH 7.8 and 37OCin the presence of cyclic AMP or cyclic overall
&P, 10 mMMgC12and lti
phosphorylation
exhibited
AMP (Fig.11 and 10m5Mcyclic phylline
concentration
aminophylline.
Under these conditions
maxima at concentrations
WP, respectively.
to 10 r&l resulted
of 10V7Mcyclic
An increase of the amino-
in a 40-50% reduction
of mernbrane-
bound [32P]phosphate at the conditions of maximm incorporation. Experimnts
performed with [r -3*P]GTP showedthat the GTP-dependent overall
phosphorylation addition
of the plam
of cyclic
Short-time kinetics
membranewas not significantly
W or cyclic
influenced by
AMP.
and the effect
of protein concentration:
The kinetic
periments were performed under standard conditions using concentrations cyclic
nucleotides which had shown optiml
lation
after
1 min incubation.
tein-bound
of overall
phosphory-
described was 2+0.5 sec. Further
were 5, 15, 60 and 600 sec. In Fig.2 the data of pro-
p*P]phosphate are given for a low concentration
teins (24ug per sample). The time courses of phosphorylation or absence of cyclic
of
The shortest incubation time which could be
achieved under the experimental conditions incubation intervals
stimlation
ex-
nucleotides
indicate
of membraneproin the presence
a rapid phosphorylation
followed by a rapid dephosphorylation and a slow rephosphorylation started between 5 and 15 sec. The stirmlation in the presence of cyclic
AMP after
of short-tim
up to 2 set which
phosphorylation
2 set incubation ranged between 70-100'10
662
Vol. 84, No. 3, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH CO/vVvtUNlCATlONS
cl
i
0
1
b Cyclic 5
1. Effects
6 nucleotide
'
of cyclic
I,
25
02
C-logB Ml *
Eal
M) Incubation
nucleotide concentration
plasma membraneproteins. under standard conditions
L
15
time
Csecl
on [32P]incorporation
into
Phosphorylation reactions were carried out using [y -J"P]ATP as radioactive precursor.
Incubation time was 1 min, the content of membraneprotein was 108 pg/ sample. Effect
of cAMP (
l
1; effect
of cGMP( o ).
2. Effect of incubation time on phosphorylation/dephosphorylation kinetics in the absence and presence of cyclic nucleotides using a low plasma membraneprotein concentration (24 ug/sample) and [T-~~P~ATP as precursor. ( l 1 without cyclic nucleotides; (V') 10-7M CAMP; (1) 10-5M cGMP.
while cyclic
GMF'showeda 20-30% inhibition
under these conditions.
5 set incubation it appeared that the presence of cyclic phosphorylation
yielding
compared to the control rephosphorylation,
After
GMPstimulated de-
only lo-40% of protein bound [3*P]phosphate when or to incubation with cyclic
after
5 to 15 set incubation,
nucleotides did not have a very specific
effect
AMP. In the phase of
the presence of cyclic on the time course of phos-
phorylation. The pattern of phosphoproteins obtained after phoresis was not significantly seen in Fig.3,
the relative
incubation is very similar
SDSpoly-acrylamide
influenced by cyclic distribution
nucleotides.
of phosphoproteins after
in the presence and absence of cyclic
663
gel electroAs can be 2 set nucleotides
BIOCHEMICAL
Vol. 84, No. 3, 1978
166
110
B
U
Z?
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
16
6
Ol 3
’
Ekctroph&c6mipmtion
hil
3. SDSpolyacrylmide
I 60
15
0
-I
Incubation
gel (5%) electrophoresis
time
of the plasm membrane
fraction, after 2 set of incubation of a low concentration membraneproteins (42pg/sample) under standard conditions. Without cyclic 4. Effect
nucleotides;
600
Csecl
( o ) 10-7M CAMP; (
l
of plasma Cotted line:
> lo-'M &ML'.
of incubation time on phosphorylation/dephosphorylation
kinetics
in the absence and presence of cyclic nucleotides using a high plasm m&x?ane protein concentration (240ug/sample) and[y-32P]ATP as precursor. (
> without cyclic
l
and differs
essentially
porated. The pattern
nucleotides;
also resembled that obtained after
by cyclic
60 set mximum stimlation of cyclic
were obtained
of membraneproteins was increased by a factor phosphorylation
AMP as well as by cyclic
10
and dephosphory-
could be no more recognized but late-phase phosphorylation
found to be stimlated
all
1 min incubation.
tima courses of phosphorylation
(24Opg per sample). The phase of short-time lation
10m5M&HP.
only in the absolute amount of [32Plphosphate incor-
Somewhatdifferent when the concentration
(V > 10W7MCAMP; (I)
was now
G&IF'(Fig.4).
After
was observed ranging between 20-708 in the presence
AMP and between IO-50% in the presence of cyclic
tin-e courses show a phase where dephosphorylation
mre rapid in the presence of cyclic
W.
predominatly occurred,
nucleotides than without.
664
Thereafter,
BIOCHEMICAL
Vol. 84, No. 3, 1978
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION The phenomenonof short-time
phosphorylation/dephosphorylation
brane proteins might represent a biologically The phosphorylation concentrations lation.
data obtained after
do not necessarily
significant
phosphorylation
is possibly achieved
slow rephosphorylation
with the linear phosphorylation kinetics
Short time phosphorylation
of membraneproteins,
phosphoprotein patterns no specific
phosphorylation
is difficult
described by a
stimulated by cyclic
nucleo-
As far as it can be judged from the
preference of one or the other membrane
The influence of high concentrations of phosphorylation
corre-
(3-16).
tides or not, appears to be not selective.
protein during short-time
phosphory-
already part of the rapid dephosphorylation
in less than one second. The phase of relatively
number of other investigators
system.
2 set incubation with low protein
time course and the maximumof short-tin-e
indicate
regulatory
correspond to maximumshort-time
These data are most likely
lates most likely
of plasma mem-
is evident.
of membraneproteins on the time course
to interpret.
Our data do not necessarily
that short-time phosphorylation/dephosphorylation
might not have
occurred in this case since the phase of rapid dephosphorylation could have already come to the end after phosphorylation
2 set incubation.
of high concentrations
stimulated in the presence of cyclic tions late-phase stinuiLation by cyclic only after
10 min incubation.
On the other hand, late-phase
of membraneproteins was found to be nucleotides.
At low protein concentra-
nucleotides attained
values
A possible explanation of this phenomenonis
offered by the assumption that increasing concentrations are speeding up the time course of short-time lation
significant
of membraneprotein
phosphorylation/dephosphory-
and rephosphorylation. REFERENCES
and E.G.Krebs (1968) J.Biol.Chem. 243, 3763-3765. 2. Kuo, J.F. and P.Greengard (1970) J.Biol.Chem. 245, 2493-2498. 3. Iabrie, F., S.Lemaire, G.Poirier, G.Pelletier and R.Boucher (1971) J.Biol. them. 246, 7311-7317. 1. Walsh, D.A., J.P.Perkins
665
vol.
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