Vol. 71, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE PHOSPHORYLATION OF RIBOSOMAL PROTEIN S6 IN BABY HAMSTER KIDNEY FIBROBLASTS
David
P. Leader,
Department
Received
Andrew
D. Rankine
and Amanda A. Coia
of Biochemistry, University of Glasgow Glasgow G12 SQQ, Scotland, U.K.
May 24,
1976 SUMMARY
Ribosomal protein 56 was extensively phosphorylated in preconfluent but not in post-confluent baby hamster kidney fibroblasts. This appears to be the first example of increased phosphorylation of S6 under physiological conditions where the cellular concentration of cyclic AMP is not elevated. The extent of the phosphorylation of S6 was also independent of alterations in the protein synthetic activity of the cells, suggesting that the biological role of this phosphorylation may be unrelated to the functional ability of the ribosomes. INTRODUCTION The extent (nomenclature stimuli.
of phosphorylation
of Sherton Cyclic
reticulocytes
(2) or rat
effects
phosphorylated differences
directly
found
in the livers or diabetic
this,
and dephosphorylated in protein
0 1976 by Academic Press, of reproduction in any form
and it in fact,
of S6 might, Despite
synthetic
Inc. reserved.
has been
ribosomes
failed
in vitro
inhibitors of 56
suggested
increase studies
(4),
increased
Although
careful
activity
966
where
the phosphorylation
may be indirect
proteins.
phosphorylation
of glucagon-treated
observed.
of
rabbit
an increased
(6) rats,
stimulate
S6
by a number
to both
(3) causes
can also
protein
can be altered
AMP have been
the phosphorylation
of certain
Copyright All rights
synthesis
31, their
(1))
liver
(51,
of cyclic
of protein
that
also
hepatectomised
concentrations
(7,
and Wool
AMP administered
of S6, a situation partial
of ribosomal
(5)
the synthesis with to reveal
(8,9),
any
and the
Vol. 71, No. 4, 1976
BIOCHEMICAL
biological
role
We thought
it
of growth
of the phosphorylation might
phosphorylation
be useful,
using
both
the cellular
concentrations
baby hamster
kidney
associated greater
with protein
of the role
cells
either synthesis,
where
of protein
of cyclic
here,
protein
concentrations
and suggest
the
unresolved. this the
conditions
synthesis
AMP (11).
presented
of ribosomal elevated
to examine
in culture,
the rates
fibroblasts,
phosphorylation
of S6 remained
therefore,
animal
can affect
increased
AND BlOPHYSfCAL RESEARCH COMMUNICATIONS
(10)
Our results demonstrate
and with that
S6 need not be of cyclic
need for
AMP or
a reappraisal
of the phosphorylation.
MATERIALS ANDMETHODS BHK21/C13 cells, an established line of hamster fibroblasts (12), were grown in rotating 80 oz. roller bottles in Eagle's medium (Glasgow modification (13)) containin 8 10% (v/v) calf serum and 10% (v/v) tryptose phosphate broth at 37 C in an atmosphere of 95%?air/5% CO2 (14). Cells were 'seeded' at a density of 2 x 10 cells per bottle in 180 ml medium, which was normally replaced after three days and every two days thereafter. The cells reached confluence after about four days but continued to grow for three or four days thereafter, albeit at a slower rate. Those cells designated pre-confluent were harvested after two or three days' growth, whereas those designated post-confluent were harvested after five to eight days' growth. In some experiments BHK21/C13/PyY cells, a variant which had been transformed by polyoma virus (15), were used.. These grew more rapidly than the parent strain, reaching confluence after about three days. 3sn experiments in which the ribosomal proteins were labelled with ( P) orthophosphate the medium was replaced by medium (50 ml) from which or i&ophosphate and tryptose phosphate broth had been excluded. ( P) orthophosphate (5mCi) was then added to two of the twenty bottles used and incubation continued for a further 3 hr before harvesting the cells. In experiments where the cells were not labelled, the medium was not replaced before harvesting the cells, unless specifically stated in the text. Cells were removed from the bottles with a rubber scraper and ribosomes isolated, essentially as described by Ascione and Arlinqhaus (16). Ribosomal subunits were prepared from these (17) Twoand the ribosomal protein extracted with acetic acid (1). dimensional gel electrophoresis, in which the first dimension contained 4% acrylamide (3.3% bisacrylamide) and 6M urea (pH 8.7) and the second dimension contained 18% acrylamide (1.4% bisacrylamide) and 6M urea (pH 4.5), was performed as previously described (18).
967
Vol. 71, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sucrose density gradient analysis of polysomes was performed by layering 1.8ml post-nuclear cell extract (16) onto a 10 - 50% linear gradient (37ml) of sucrose in a solution containing 5OmM Tris2oOm~ KCl, 5mM MgC12 and centrifuging for 2 hr at HCl (pH 7.6), 82,5OCg in a SW27 rotor of a Beckman ultracentrifuge at 2OC. The gradients were then pumped through the flow-cell of a Gilford Model 240 recording spectrophotometer and analysed at 260nm. The concentration of cyclic AMP in BHK cells was determined by a modification (19) of the protein-binding competition assay of by the Radiochemical Centre, Gilman (201, using a kit supplied Amersham, Bucks, U.K. A trichloroacetic acid extract of the cells was prepared for assay by ether extraction, followed by elution with 2N formic acid from a Dowex-1 (formate) column (21). The values obtained were corrected for the recovery of (3H) cyclic AMP added to the original extract and expressed in terms of the total cellular protein, determined by a modification of the Lowry method (22).
RESULTS AND DISCUSSION The phosphorylation formation
of derivatives
phosphoryl
groups,
be visualised protein
in
of
the protein
the stained
gels
to two-dimensional on the basis
(3,
24).
5, 23,
in Fig. upward
This
1 where from,
associated
the parent
of unlabelled
this
approach
'tail'
two.problems
associated
the effect
of the state (251,
after
for
'tail' II gross
by examining proteins.
the use of
that
of,
involving
BHK cells
and slightly
ascites changes stained
We decided
the phosphorylation
and the fact
the
dimension
was far
in Krebs
of cellular
subjecting
to the radioactivity
of detecting
with
can
pre-confluent
to the left
the
up to five of these
the first
This
merely
involves
in systems
S6 corresponds
ribosomal
to studying
in
illustrated
observed
of 56,
gels
phosphate
is
the possibility
phosphorylation
obtained
of charge
the protein.
we had previously
indicated
S6
containing
electrophoresis
an anodic
with
protein
and the more phosphorylated
separation
than
of ribosomal
growth
as it
cells
(23)
and
in the two-dimensional to adopt circumvented
( 32 P) orthophosphate: on the uptake
the phosphate-deficient
968
more extensive
of medium
BIOCHEMICAL
Vol. 71, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Phosphorylation of ribosomal protein 56 in pre-confluent Figure 1. The upper frame (i) BHK cells incubated with t3*P) orthophosphate. shows a stained gel and the lower frame (ii) the relevant area of the corresponding autoradiograph following two-dimensional gel electrophoresis of ribosomal protein (2OOug) from the 405 subunit. The arrow and parenthesis marks, indicating the beginning and end of the in both frames. tail of S6, are in identical positions
we used to label
the
being
(see Fig.
considered
cells
When we examined of S6 in unlabelled much lower was somewhat concentrations
altered
the extent
in post-confluent
of growing
of protein
synthesis
3, below).
BHK cells
surprising
the condition
of the phosphorylated 2) it
(Fig. than
in view cells
was clear
in pre-confluent of reports are
969
generally
that
derivatives that cells.
the lower
cyclic than
this
was This
AMP those
of
Vol. 71, No. 4, 1976
BIOCHEMICAL
+ O-
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lst Dimension c3
0)
The phosphorylation of ribosomal protein S6 in pre-confluent Figure 2. The frames show stained two-dimensional BHK cells. and post-confluent gels of ribosomal protein from the 40s subunits of unlabelled (i.) The parentheses pre-confluent, and (ii) post-confluent BHK cells. delineate the visible extent of protein S6 and its derivatives and as reference the positions of proteins 514, SlO and S19 are indicated points.
resting
cells.
of this
nucleotide
We thus in our
felt
it
necessary
own cells
to measure
and found
970
that
the
there
levels was only
Vol. 71, No. 4, 1976
a small
BIOCHEMICAL
difference
pre-confluent protein,
in
respectively)
et al
which
of protein
in growing
and resting
arrested,
in contrast important
pre-confluent
knowledge,
It
that
the
whose
time
and those
pmoles
cyclic
This
study.
AMP in
there
has been
independently
completely
in this
is,
mg
is
had been
and yet
of Rudland
AMP per
respectively)
of cyclic
such an increase
conditions,
statistically
growth
S6.
the
and 2.4 pmole/mg
cells
elevated
protein
AMP in
results
BHK cells,
concentration
is not
of ribosomal the first
of cyclic
these
to the post-confluent
is
physiological
fact,
of 7.6 and 13.2
use of cells
BHK cells
phosphorylation
(1.8
in
between
values
due to their
under
was not,
(who found
probably
of cyclic
BHK cells
The difference
(11)
What is
concentration
and post-confluent
significant.
is
extensive
to our observed
of increased
levels
AMP. seemed possible
phosphorylation
to different
Because
the
cellular
that
the difference
of S6 in pre-confluent
related
into
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
comparative protein
rates
of protein
incorporation presents
between
and post-confluent synthesis
in
of radioactive
difficulties
the cells
these amino
was
cells. acids
of interpretation
Figure 3. Sucrose density gradient analysis of polysomes and monosomes from BHK cells. Sedimentation was from right to left, as described in the text. (i) pre-confluent cells (2 days of growth) (ii) post-confluent cells (7 days of growth) (iii) post-confluent cells incubated 1 hr in fresh, complete, medium (iv) pre-confluent cells incubated 3 hr in medium lacking tryptose phosphate broth.
971
BIOCHEMICAL
Vol. 71, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 4. Effect of fresh medium on the phosphorylation of ribosomal The frames show stained protein S6 in post-confluent BHK cells. two-dimensional gels of ribosomal proteins from the 405 subunits of unlabelled post-confluent BHK cells (i) No addition of fresh medium (ii) Fresh medium added 1 hr before harvesting before harvesting cells. cells.
similar
to those
polysomes
with
to monosomes
synthesis.
Fig.
polysomes
was lower
cells,
or'chophosphate,
but
that
3,
cells
this
related supported
41,
this
that
of protein
by other
considerations. cells
results
in a decrease
However
the pre-confluent
2, i.
with in
Finally,
synthesis.
of fresh
This
Thus,
not
of Fig.
conclusion
1 were
phosphate
and post-confluent
(Fig.
labelled
than
was
of the medium
tryptose
difference
972
of S6 was not
of polysomes
phosphorylated
a similar
medium was
medium did
replacement
lacking
the proportion
of ~6 was seen in pre-confluent
in pre-confluent
of S6 in post-confluent
a medium
S6 no less
of
when fresh
addition
of
of protein
the phosphorylation
cells
ratio
the proportion than
of phosphorylation
to the rate
a medium but have
that
was abolished
However
suggesting
of pre-confluent
of Fig.
shows
difference
used the
of the rates
in the post-confluent
the extent (Fig.
as an indication (i)-(iii),
added to the former. increase
we have
3, iv).
in
just
the pre-confluent
in extent cells
broth
such cells
of phosphorylation of the more
BIOCHEMICAL
Vol. 71, No. 4, 1976
rapidly
growing,
polyoma-transformed,
Our findings S6 can occur
that
without
we have (5)
no direct
can be reconciled
the nucleus,
protein but
certain
is
normally to If
this
either
a higher
proportion
lower
cytoplasmic
increased
are required
we are
phosphoprotein
Although
attracted
to the
be important
ones
the
our
after
leaving
rephosphorylation
the greater
BHK cells synthesised
phosphatase
idea
(2 - 7) by assuming
(non-functional) so,
for
is because
dephosphorylated
were
phosphorylation
could
reflect
ribosomes activity
or a
than
in
cells.
However
is
of newly
of this
cyclic
into
the cytoplasm.
This
previous
of 56 in pre-confluent
of S6, for
in
the nucleolus.
phosphorylation
post-confluent
synthesis role
instead,
conditions.
protein
call
might,
susceptible
of ribosomal
of protein
it,
S6 is
shown).
of cellular
to support
with
(not
of the concentration
of ribosomes
in
RESEARCH COMMUNICATIONS
phosphorylation
the biological
evidence
of ribosomes
ribosomal
under
that
the phosphorylation
formation
that
ideas
to the function
that
results
extensive
of the rate
previous
is related
PyY line
an elevation
AMP and independently question
AND BIOPHYSICAL
there
example
are other
conceivable
in preventing
in resting
cells
to distinguish
roles
the degradation Clearly,
(26). between
these
for
the phosphorylation
of ribosomes, further
and other
which
experiments possibilities.
ACKNOWLEDGEMENTS We thank Mrs. J. McWattie for Drs. R.L.P. Adams, J.P. Durham and This work was supported by a grant the Cancer Research Campaign and an to A.D.R.
able technical assistance and W.S. Stevely for helpful discussion. to Professor R.M.S. Smellie from M.R.C. postgraduate training grant
REFERENCES 1. 2.
Sherton,C.C. and Woo1,I.G. (1972) J.Biol.Chem. 247, Cawthon,M.L., Bitte,L.F., Krystosek,A. and Kabat,D. J.Biol.Chem. 249, 275-278.
973
4460-4467. (1974).
Vol. 71, No. 4, 1976
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Gressner,A.M. and Wo01.1.G. (1974). Biochem.Biophys.Res.Commun. 60, 1482-1489. Blat,C. and Loeb,J.E. (1971). FEBS Lett. 18, 124-126. Gressner,A.M. and Woo1,I.G. (1974). J.Biol.Chem. 249, 6917-6925. Gressner,A.M. and Woo1,I.G. (1976). Nature, 259, 148-150. Kabat,D. (1970). Biochemistry, 9, 4160-4175. Eil,C. and Woo1,I.G. (1973). J.Biol.Chem. 248, 5130-5136. Krystosek,A., Bitte,L.F., Cawthon,M.L. and Kabat,D. (1974). Ribosomes, pp 855-870, Cold Spring Harbor Press, New York. van Venrooij,W.J.W., Henshaw,E.C. and Hirsch,C.A. (1970). J.Biol.Chem. 245, 5947-5953. Rudland,P.S., Seeley,M. and Seifert,W. (1974). Nature, 259, 148-150. Macpherson,I.A. and Stoker,M.G.P. (1962). Virology, 16, 147-151. Busby,D.W.G., House, W. and McDonald, J.R. (1964). Virological Technique, Churchill, London. Lab. Practice, 14, 594-595. House,W. and Wildy,P. (1965). Stoker,M.G.P. and Macpherson,I.A. (1964). Nature, 203, 1355-1357. Ascione,R. and Arlinghaus,R.B. (1970). Biochim.Biophys.Acta, 204, 478-488. Leader,D.P. and Woo1,I.G. (1972). Biochim.Biophys. Acta, 262, 360-370. Leader,D.P. (1975). Biochem. J. 152, 373-378. Tovey,K.C., Oldham,K.G. and Whelan,J.A.M. (1974). Clin.Chim.Acta, 56, 221-234. Gilman,A.G. (1970). Proc.Natl.Acad.Sci. U.S. 67, 305-312. Murad,F., Manganiello,V. and Vaughan,M. (1971). Proc.Natl.Acad.Sci. U.S. 68, 736-739. Lowry,O.H., Rosenbrough,N.J., Farr,A.L. and Randal1,R.J. (1951). J.Biol.Chem. 193, 265-275. Rankine,A.D. and Leader,D.P. (1975). FEBS Lett. 52, 284-287, and Porter,G.G. (1976). Biochemistry 15, 610-616. Traugh,J.A. Cunningham,D.D. and Pardee,A.B. (1969). Proc.Natl.Acad.Sci. U.S. 64, 1049-1056. Abelson,H.T., Johnson,L.F., Penman,S. and Green,H. (1974). Cell, 1, 161-165.
974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE PHOSPHORYLATION OF RIBOSOMAL PROTEIN S6 IN BABY HAMSTER KIDNEY FIBROBLASTS
David
P. Leader,
Department
Received
Andrew
D. Rankine
and Amanda A. Coia
of Biochemistry, University of Glasgow Glasgow G12 SQQ, Scotland, U.K.
May 24,
1976 SUMMARY
Ribosomal protein 56 was extensively phosphorylated in preconfluent but not in post-confluent baby hamster kidney fibroblasts. This appears to be the first example of increased phosphorylation of S6 under physiological conditions where the cellular concentration of cyclic AMP is not elevated. The extent of the phosphorylation of S6 was also independent of alterations in the protein synthetic activity of the cells, suggesting that the biological role of this phosphorylation may be unrelated to the functional ability of the ribosomes. INTRODUCTION The extent (nomenclature stimuli.
of phosphorylation
of Sherton Cyclic
reticulocytes
(2) or rat
effects
phosphorylated differences
directly
found
in the livers or diabetic
this,
and dephosphorylated in protein
0 1976 by Academic Press, of reproduction in any form
and it in fact,
of S6 might, Despite
synthetic
Inc. reserved.
has been
ribosomes
failed
in vitro
inhibitors of 56
suggested
increase studies
(4),
increased
Although
careful
activity
966
where
the phosphorylation
may be indirect
proteins.
phosphorylation
of glucagon-treated
observed.
of
rabbit
an increased
(6) rats,
stimulate
S6
by a number
to both
(3) causes
can also
protein
can be altered
AMP have been
the phosphorylation
of certain
Copyright All rights
synthesis
31, their
(1))
liver
(51,
of cyclic
of protein
that
also
hepatectomised
concentrations
(7,
and Wool
AMP administered
of S6, a situation partial
of ribosomal
(5)
the synthesis with to reveal
(8,9),
any
and the
Vol. 71, No. 4, 1976
BIOCHEMICAL
biological
role
We thought
it
of growth
of the phosphorylation might
phosphorylation
be useful,
using
both
the cellular
concentrations
baby hamster
kidney
associated greater
with protein
of the role
cells
either synthesis,
where
of protein
of cyclic
here,
protein
concentrations
and suggest
the
unresolved. this the
conditions
synthesis
AMP (11).
presented
of ribosomal elevated
to examine
in culture,
the rates
fibroblasts,
phosphorylation
of S6 remained
therefore,
animal
can affect
increased
AND BlOPHYSfCAL RESEARCH COMMUNICATIONS
(10)
Our results demonstrate
and with that
S6 need not be of cyclic
need for
AMP or
a reappraisal
of the phosphorylation.
MATERIALS ANDMETHODS BHK21/C13 cells, an established line of hamster fibroblasts (12), were grown in rotating 80 oz. roller bottles in Eagle's medium (Glasgow modification (13)) containin 8 10% (v/v) calf serum and 10% (v/v) tryptose phosphate broth at 37 C in an atmosphere of 95%?air/5% CO2 (14). Cells were 'seeded' at a density of 2 x 10 cells per bottle in 180 ml medium, which was normally replaced after three days and every two days thereafter. The cells reached confluence after about four days but continued to grow for three or four days thereafter, albeit at a slower rate. Those cells designated pre-confluent were harvested after two or three days' growth, whereas those designated post-confluent were harvested after five to eight days' growth. In some experiments BHK21/C13/PyY cells, a variant which had been transformed by polyoma virus (15), were used.. These grew more rapidly than the parent strain, reaching confluence after about three days. 3sn experiments in which the ribosomal proteins were labelled with ( P) orthophosphate the medium was replaced by medium (50 ml) from which or i&ophosphate and tryptose phosphate broth had been excluded. ( P) orthophosphate (5mCi) was then added to two of the twenty bottles used and incubation continued for a further 3 hr before harvesting the cells. In experiments where the cells were not labelled, the medium was not replaced before harvesting the cells, unless specifically stated in the text. Cells were removed from the bottles with a rubber scraper and ribosomes isolated, essentially as described by Ascione and Arlinqhaus (16). Ribosomal subunits were prepared from these (17) Twoand the ribosomal protein extracted with acetic acid (1). dimensional gel electrophoresis, in which the first dimension contained 4% acrylamide (3.3% bisacrylamide) and 6M urea (pH 8.7) and the second dimension contained 18% acrylamide (1.4% bisacrylamide) and 6M urea (pH 4.5), was performed as previously described (18).
967
Vol. 71, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sucrose density gradient analysis of polysomes was performed by layering 1.8ml post-nuclear cell extract (16) onto a 10 - 50% linear gradient (37ml) of sucrose in a solution containing 5OmM Tris2oOm~ KCl, 5mM MgC12 and centrifuging for 2 hr at HCl (pH 7.6), 82,5OCg in a SW27 rotor of a Beckman ultracentrifuge at 2OC. The gradients were then pumped through the flow-cell of a Gilford Model 240 recording spectrophotometer and analysed at 260nm. The concentration of cyclic AMP in BHK cells was determined by a modification (19) of the protein-binding competition assay of by the Radiochemical Centre, Gilman (201, using a kit supplied Amersham, Bucks, U.K. A trichloroacetic acid extract of the cells was prepared for assay by ether extraction, followed by elution with 2N formic acid from a Dowex-1 (formate) column (21). The values obtained were corrected for the recovery of (3H) cyclic AMP added to the original extract and expressed in terms of the total cellular protein, determined by a modification of the Lowry method (22).
RESULTS AND DISCUSSION The phosphorylation formation
of derivatives
phosphoryl
groups,
be visualised protein
in
of
the protein
the stained
gels
to two-dimensional on the basis
(3,
24).
5, 23,
in Fig. upward
This
1 where from,
associated
the parent
of unlabelled
this
approach
'tail'
two.problems
associated
the effect
of the state (251,
after
for
'tail' II gross
by examining proteins.
the use of
that
of,
involving
BHK cells
and slightly
ascites changes stained
We decided
the phosphorylation
and the fact
the
dimension
was far
in Krebs
of cellular
subjecting
to the radioactivity
of detecting
with
can
pre-confluent
to the left
the
up to five of these
the first
This
merely
involves
in systems
S6 corresponds
ribosomal
to studying
in
illustrated
observed
of 56,
gels
phosphate
is
the possibility
phosphorylation
obtained
of charge
the protein.
we had previously
indicated
S6
containing
electrophoresis
an anodic
with
protein
and the more phosphorylated
separation
than
of ribosomal
growth
as it
cells
(23)
and
in the two-dimensional to adopt circumvented
( 32 P) orthophosphate: on the uptake
the phosphate-deficient
968
more extensive
of medium
BIOCHEMICAL
Vol. 71, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Phosphorylation of ribosomal protein 56 in pre-confluent Figure 1. The upper frame (i) BHK cells incubated with t3*P) orthophosphate. shows a stained gel and the lower frame (ii) the relevant area of the corresponding autoradiograph following two-dimensional gel electrophoresis of ribosomal protein (2OOug) from the 405 subunit. The arrow and parenthesis marks, indicating the beginning and end of the in both frames. tail of S6, are in identical positions
we used to label
the
being
(see Fig.
considered
cells
When we examined of S6 in unlabelled much lower was somewhat concentrations
altered
the extent
in post-confluent
of growing
of protein
synthesis
3, below).
BHK cells
surprising
the condition
of the phosphorylated 2) it
(Fig. than
in view cells
was clear
in pre-confluent of reports are
969
generally
that
derivatives that cells.
the lower
cyclic than
this
was This
AMP those
of
Vol. 71, No. 4, 1976
BIOCHEMICAL
+ O-
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lst Dimension c3
0)
The phosphorylation of ribosomal protein S6 in pre-confluent Figure 2. The frames show stained two-dimensional BHK cells. and post-confluent gels of ribosomal protein from the 40s subunits of unlabelled (i.) The parentheses pre-confluent, and (ii) post-confluent BHK cells. delineate the visible extent of protein S6 and its derivatives and as reference the positions of proteins 514, SlO and S19 are indicated points.
resting
cells.
of this
nucleotide
We thus in our
felt
it
necessary
own cells
to measure
and found
970
that
the
there
levels was only
Vol. 71, No. 4, 1976
a small
BIOCHEMICAL
difference
pre-confluent protein,
in
respectively)
et al
which
of protein
in growing
and resting
arrested,
in contrast important
pre-confluent
knowledge,
It
that
the
whose
time
and those
pmoles
cyclic
This
study.
AMP in
there
has been
independently
completely
in this
is,
mg
is
had been
and yet
of Rudland
AMP per
respectively)
of cyclic
such an increase
conditions,
statistically
growth
S6.
the
and 2.4 pmole/mg
cells
elevated
protein
AMP in
results
BHK cells,
concentration
is not
of ribosomal the first
of cyclic
these
to the post-confluent
is
physiological
fact,
of 7.6 and 13.2
use of cells
BHK cells
phosphorylation
(1.8
in
between
values
due to their
under
was not,
(who found
probably
of cyclic
BHK cells
The difference
(11)
What is
concentration
and post-confluent
significant.
is
extensive
to our observed
of increased
levels
AMP. seemed possible
phosphorylation
to different
Because
the
cellular
that
the difference
of S6 in pre-confluent
related
into
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
comparative protein
rates
of protein
incorporation presents
between
and post-confluent synthesis
in
of radioactive
difficulties
the cells
these amino
was
cells. acids
of interpretation
Figure 3. Sucrose density gradient analysis of polysomes and monosomes from BHK cells. Sedimentation was from right to left, as described in the text. (i) pre-confluent cells (2 days of growth) (ii) post-confluent cells (7 days of growth) (iii) post-confluent cells incubated 1 hr in fresh, complete, medium (iv) pre-confluent cells incubated 3 hr in medium lacking tryptose phosphate broth.
971
BIOCHEMICAL
Vol. 71, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 4. Effect of fresh medium on the phosphorylation of ribosomal The frames show stained protein S6 in post-confluent BHK cells. two-dimensional gels of ribosomal proteins from the 405 subunits of unlabelled post-confluent BHK cells (i) No addition of fresh medium (ii) Fresh medium added 1 hr before harvesting before harvesting cells. cells.
similar
to those
polysomes
with
to monosomes
synthesis.
Fig.
polysomes
was lower
cells,
or'chophosphate,
but
that
3,
cells
this
related supported
41,
this
that
of protein
by other
considerations. cells
results
in a decrease
However
the pre-confluent
2, i.
with in
Finally,
synthesis.
of fresh
This
Thus,
not
of Fig.
conclusion
1 were
phosphate
and post-confluent
(Fig.
labelled
than
was
of the medium
tryptose
difference
972
of S6 was not
of polysomes
phosphorylated
a similar
medium was
medium did
replacement
lacking
the proportion
of ~6 was seen in pre-confluent
in pre-confluent
of S6 in post-confluent
a medium
S6 no less
of
when fresh
addition
of
of protein
the phosphorylation
cells
ratio
the proportion than
of phosphorylation
to the rate
a medium but have
that
was abolished
However
suggesting
of pre-confluent
of Fig.
shows
difference
used the
of the rates
in the post-confluent
the extent (Fig.
as an indication (i)-(iii),
added to the former. increase
we have
3, iv).
in
just
the pre-confluent
in extent cells
broth
such cells
of phosphorylation of the more
BIOCHEMICAL
Vol. 71, No. 4, 1976
rapidly
growing,
polyoma-transformed,
Our findings S6 can occur
that
without
we have (5)
no direct
can be reconciled
the nucleus,
protein but
certain
is
normally to If
this
either
a higher
proportion
lower
cytoplasmic
increased
are required
we are
phosphoprotein
Although
attracted
to the
be important
ones
the
our
after
leaving
rephosphorylation
the greater
BHK cells synthesised
phosphatase
idea
(2 - 7) by assuming
(non-functional) so,
for
is because
dephosphorylated
were
phosphorylation
could
reflect
ribosomes activity
or a
than
in
cells.
However
is
of newly
of this
cyclic
into
the cytoplasm.
This
previous
of 56 in pre-confluent
of S6, for
in
the nucleolus.
phosphorylation
post-confluent
synthesis role
instead,
conditions.
protein
call
might,
susceptible
of ribosomal
of protein
it,
S6 is
shown).
of cellular
to support
with
(not
of the concentration
of ribosomes
in
RESEARCH COMMUNICATIONS
phosphorylation
the biological
evidence
of ribosomes
ribosomal
under
that
the phosphorylation
formation
that
ideas
to the function
that
results
extensive
of the rate
previous
is related
PyY line
an elevation
AMP and independently question
AND BIOPHYSICAL
there
example
are other
conceivable
in preventing
in resting
cells
to distinguish
roles
the degradation Clearly,
(26). between
these
for
the phosphorylation
of ribosomes, further
and other
which
experiments possibilities.
ACKNOWLEDGEMENTS We thank Mrs. J. McWattie for Drs. R.L.P. Adams, J.P. Durham and This work was supported by a grant the Cancer Research Campaign and an to A.D.R.
able technical assistance and W.S. Stevely for helpful discussion. to Professor R.M.S. Smellie from M.R.C. postgraduate training grant
REFERENCES 1. 2.
Sherton,C.C. and Woo1,I.G. (1972) J.Biol.Chem. 247, Cawthon,M.L., Bitte,L.F., Krystosek,A. and Kabat,D. J.Biol.Chem. 249, 275-278.
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Vol. 71, No. 4, 1976
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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