T H E r
11:’ 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
OF BIOI.OGICAL CliEMlSTRY
Vol. 266. No. 33, Issue of November 25. pp. 22770-22775, 1991
Printed in U.S.A.
Mitogen-activated 70K S6 Kinase
IDENTIFICATION OF IN VITRO 40 S RIBOSOMAL S6 PHOSPHORYLATION SITES*
(Received for publication, June 24, 1991)
Stefan0 Ferrari, H. Regina Bandi, Jan Hofsteenge, Bernd M. BussianS, and George Thomas5
From the Friedrich Miescher Institute. Postfach 2543. CH-4002 Basel, Switzerland and the SFachinformationszentrum,
Ciba-Geigy AG, CH-4002 Basel, Switzerland
Recently we purified and cloned the mitogen/onco-
gene-activated M, 70,000 (70K) S6 kinase from the
livers of rats treated with cycloheximide (Kozma, S.
C., Lane, H. A., Ferrari, S., Luther, H., Siegmann, M.,
and Thomas, G. (1989) EMBO J. 8, 4125-4132;
Kozma, S. C., Ferrari, S., Bassand, P., Siegmann, M.,
Totty, N., and Thomas, G. (1990) Proc. Nutl. Acud.
Sci. U. S. A. 87, 7365-7369). Prior to determining the
ability of this kinase to phosphorylate the same sites
observed in S6 in vivo, we established the effects of
different cations and autophosphorylation on kinase
activity. The results show that the 70K S6 kinase is
dependent on Mg2+ for activity and that this require-
ment cannot be substituted for by MnZ+. Furthermore,
50-fold lower concentrations of Mn2+ block the effect
of Mg”+ on the kinase. This effect
but can be substituted for by a number of cations, with
Zn“+ being the most potent inhibitor, IC5o = 2 p
the presence of optimum Mg2+ concentrations the en-
zyme incorporates an average of 1.2 mol of phosphate/
mol of kinase and an average of 3.7 mol of phosphate/
mol of S6. The autophosphorylation reaction appears
to be intramolecular and leads to a 25% reduction in
kinase activity toward S6. In the case of S6 all of the
sites of phosphorylation are found to reside in a 19-
amino acid peptide at the carboxyl end of the protein.
Four of these sites have been identified
s e r 2 3 6 , SerZ4O, and Ser244, equivalent to four
is not limited to Mn2+
of the five
sites previously observed
steenge, J., and Thomas, G. (1988) J. Biol. Chem. 263,
11473-11477). A fifth mole of phosphate is incorpo-
rated at low stoichiometry into the peptide, but
amino acid which is phosphorylated cannot be une-
quivocally assigned. The low level of phosphorylation
of the fifth site in vitro is discussed with regard to
known results and to
a potential three-dimensional
model for the carboxyl terminus
(Krieg, J., Hof-
By a number of indirect methods ribosomal protein S6 has
been mapped to the region of the 40 S subunit which directly
associates with the 60 S subunit at the codon-anticodon
interaction site (1, 2). Consistent with this finding, multiple
phosphorylation of S6 is an apparent prerequisite
creased rates of initiation of protein synthesis and subsequent
cell growth in response to numerous mitogens and
factors (2,3). This functional response appears to be mediated
by the more highly phosphorylated forms of S6 (4-6).
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
8 To whom correspondence should be addressed.
To identify the sites of phosphorylation, S6 was initially
isolated from the livers of rats treated with cycloheximide, a
drug known to induce many of the early mitogenic responses
(7-lo), including S6 phosphorylation (11). Direct sequencing
of this material showed that these sites reside in 5 of 7 serines
at the carboxyl terminus of the protein (12). Earlier peptide
mapping data further indicated that S6 phosphorylation is
ordered (13, 14),
although this has yet to be established at the
amino acid sequence level.
A number of kinases have been implicated in
phorylation of S6 (2),
but to establish their biological impor-
tance it must be shown that they faithfully phosphorylate the
same sites in S6 as observed i n vivo (12). Identification of
such enzymes would facilitate studies aimed at clarifying the
importance of individual phosphorylation sites in the activa-
tion of protein synthesis as well as uncovering the mecha-
nisms required for controlling this event.
The putative kinase activity responsible for phosphorylat-
ing S6 in Swiss 3T3 cells was first detected in extracts
quiescent cells stimulated to proliferate
growth factor (15). To recover full kinase activity from such
extracts required the presence of phosphatase inhibitors, in-
dicating that the kinase itself was directly
phosphorylation (15). Purification of the kinase to homoge-
neity revealed that it is a single polypeptide of M, 70,000 (16)
and that it is directly activated by serine/threonine phos-
phorylation (17). We have shown that at least two intracel-
lular signaling pathways converge on the activation of the
kinase (18, 19),
both of which appear distinct from
the microtubule-associated protein 2 kinase pathway (20, 21).
Recently, sufficient amounts of the 70K kinase were obtained
from the livers of cycloheximide-treated rats (11, 22) to allow
both protein sequencing and molecular cloning of the enzyme
(23). It should be noted that another cDNA clone has been
isolated which is identical in the coding region to the clone
above except for a single amino acid difference and a 23-
amino acid extension at the amino terminus (24). Sequencing
of both clones revealed that the 70K S6 kinase is distinct
from the M, 92,000 S6
kinase I1 first described in extracts of
unfertilized Xenopus eggs (25, 26) and more recently in mi-
togenically stimulated avian and mouse cells (27-29) and that
it represents a new member of the second messenger family
of protein kinases (30).
Most of the studies with S6 kinases described to date have
been based on the ability of the kinase to phosphorylate either
40 S ribosomes containing S6 (15, 25, 27, 28)
eight-amino acid peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala,
which contains two of the sites of phosphorylation (20). Here
we have characterized a number of the properties of the S6
kinase with regard to cation requirements and autophosphor-
ylation. Having established optimal conditions for S6 phos-
phorylation i n uitro we have used two-dimensional polyacryl-
by serum or epidermal
or a synthetic
In Vitro S6 Phosphorylation Sites
n m (mln)
FIG. 5. Reverse phase HPLC purification of S6 peptides.
The endoproteinase Lys-C digest was made 0.1% in trifluoroacetic
acid and applied to a CIS narrow-bore glass-lined column. The column
was developed at a flow rate of 0.2 ml/min with a gradient of
acetonitrile from 0 to 50%. The protein was monitored at 214 nm,
and “’P was monitored by Cerenkov counting.
S6 Lys-C m
Kd P I
6.55 - -
3 . 5
FIG. 6. A, SDS-Tricine polyacrylamide gel. An aliquot of the peak
fraction of ‘”P-labeled S6 peptide eluting at 17.5% acetonitrile was
analyzed on SDS-Tricine polyacrylamide gel. Lane 1, undigested S6;
lane 2, Lys-C digested S6. B, isoelectric focusing gel. An aliquot of
the peak of radioactivity eluted from Cla was analyzed on a Pharmacia
PhastGel IEF 3-9.
amide gel electrophoresis as well as one-dimensional poly-
acrylamide isoelectric focussing to measure the extent of the
reaction. More importantly, we then used this material to
identify the sites of S6 phosphorylation by direct Edman
sequencing following conversion of the phosphorylated serines
to the stable analogue S-ethylcysteine.
EXPERIMENTAL PROCEDURES AND RESULTS’
Isolation of “PS6 Peptides-Recent studies have put into
question the in vivo sites of S6 phosphorylation and the kinase
responsible for controlling this event (41), prompting us to
’ Portions of this paper (including “Experimental Procedures,” part
of “Results,” Figs. 1-4, and Table I) are presented in miniprint at the
end of this paper. Miniprint is easily read with the aid of a standard
magnifying glass. Full size photocopies are included in the microfilm
edition of the Journal that is available from Waverly Press.
Amino acid released at each cycle of the Edman sequencing reaction
of the endoproteinase Lys-C S6 peptide
Cycle 1st peptide
determine the sites of phosphorylation catalyzed by the 70K
S6 kinase. Employing the assay conditions described above,
the 70K S6 kinase was capable of incorporating up to 3.7 mol
of phosphate/mol of S6 (see “Experimental Procedures”).
Two-dimensional polyacrylamide gel analysis revealed that
most of S6 migrated in the position of derivative d, containing
4 mol of phosphate, with a smaller amount migrating in the
position of derivative c and possibly e, containing 3 and 5 mol
of phosphate, respectively (see Refs. 4 and 42, data not
shown). To identify these sites S6 was purified as previously
described (E), except for two modifications (see “Experimen-
tal Procedures”). First, the protein was digested with endo-
proteinase Lys-C rather than cyanogen bromide. This led to
a substantially shorter peptide than that generated by cyan-
ogen bromide (see below), which contained all of the sites of
phosphorylation. The shorter peptide led to higher recoveries
of individual amino acids during Edman sequencing. It should
also be noted that no cleavage occurred at Lys243, possibly due
to the phosphorylation of Ser244 (see below). The second
modification was the use of a totally inert HPLC2 system for
the isolation of peptides (see “Experimental Procedures”).
This system circumvents large losses of phosphopeptides
through their interaction with metal ions, allowing the isola-
tion of picomolar amounts of phosphopeptides. The endopro-
teinase Lys-C digest yielded a single radioactive peak which
eluted from the C18 column at 17.5% acetonitrile (Fig. 5).
Approximately 80% of the applied Cerenkov counts were
recovered from the column, and all were present in this peak.
Next, a portion of this material was analyzed on an SDS-
Tricine polyacrylamide gel (33) followed by autoradiography.
As can be seen in Fig. 6A, a single phosphopeptide is obtained
with M, 2,400, which migrates between two synthetic S6
peptides of M, 2,000 (S62RP-249)
substantially slower migration of the in uitro labeled 19-amino
acid peptide derived from 40 S ribosomes uersus the synthetic
18-amino acid peptide is due to its higher extent of phos-
phorylation (data not shown) and probably reflects poor in-
and M, 3,500 (S6218-249).
* The abbreviations used are: HPLC, high performance liquid chro-
matography; MOPS, 4-morpholinepropanesulfonic acid; DTT, DL-
dithiothreitol; pNPP, 4-nitrophenyl phosphate; SDS, sodium dodecyl
sulfate; TFA, trifluoroacetic acid; Tricine, N-[2-hydroxy-l,l-bis(hy-
droxymethyl)ethyl]glycine; PTH, phenylthiohydantoin; EGTA, [eth-
In Vitro S6 Phosphorylation Sites
FIG. 7. Model of carboxyl terminus of S6. The last 20 amino acids of S6 were lined up by QUANTA
(QUANTA 3.0, CHARMm, Polygen Corp,) as a standard N helix and subsequently energy minimized by
CHARMm (50) (constant dielectricurn, 8-A nonbonded cutoff, 2000 steps of adopted basis Newton-Raphson
minimization). Residues are labeled at their C,, position. N, blue; 0, reg C, white; Ser-P, orange. Panels A and E
represent a view of the peptide which can be visualized in stereo.
teraction of SDS with phosphoryl groups.
Identification of Phosphorylated Serine Residues-To iden-
tify the sites of phosphorylation in the endoproteinase Lys-C
peptide, the phosphoserines were converted to the stable
amino acid analogue S-ethylcysteine and the peptide was
sequenced as previously described (Ref. 32 and "Experimental
Procedures"). The initial amount of peptide was 55 pmol and
the repetitive yield was 94%. The results of each cycle of the
Edman degradation are listed in Table 1 1 . As can be seen, a
second peptide copurified with the radioactively labeled S6
peptide. The peptide corresponds to a protein fragment from
amino acids 15-23 of the S6 sequence. PTH-S-ethylcysteine
could be detected at cycles 5,6,10, and 14, sites equivalent to
4 of the 5 serines observed in vivo (12). Contrary to the in
vivo phosphorylated S6, cycle 17 showed no increase in PTH-
S-ethylcysteine. However, unlike the preceding cycles, 15 and
16, the amount of PTH-S-ethylcysteine did not drop substan-
tially in cycle 17, indicating that SeP7 may have been phos-
phorylated at very low stoichiometry (data not shown). To
test this possibility, a portion of the peak fraction of the
HPLC-purified endoproteinase Lys-C S6 peptide (Fig. 5) was
analyzed on isoelectric focusing gels (Fig. 6B). The results
reveal a major phosphopeptide, containing 4 mol of phosphate
with an isoelectric point of 5.2, and two minor forms, repre-
senting 3 and 5 mol of phosphate with isoelectric points of 6
and 4.5, respectively, values almost equivalent to those ex-
pected for the endoproteinase Lys-C S6 peptide containing 3
(PI = 6-30), 4 (PI = 5.55), and 5 (PI = 4.78) mol of phosphate.
This last result demonstrates the existence of a fifth phos-
phorylation site but does not allow us to designate which of
the possible remaining three serines is phosphorylated.
A number of kinases have been implicated in the phos-
phorylation 'of S6, but in only a few instances has attention
been paid to whether the same sites are phosphorylated in
vitro as observed in vivo (2). Indeed, the protein contains a
number of consensus sequences for such ubiquitously ex-
pressed protein kinases as the cyclic AMP-dependent protein
kinase (43) and the protein kinase C family (44). Thus, it is
an essential prerequisite in studying the mechanism and
kinases which control this response to first establish the in
vivo sites of phosphorylation. From studies carried out in vitro
and in vivo it was first pointed out by Wettenhall and col-
leagues (45) that eight potential multiple sites for S6 phos-
phorylation resided in a 17-amino acid tryptic peptide of the
protein. Subsequently, we exploited the use of cycloheximide
injection of rats to obtain sufficient amounts of highly phos-
phorylated S6 to demonstrate (i) that the peptide described
by Wettenhall contained all the sites of S6 phosphorylation,
(ii) that this peptide resided at the carboxyl terminus of S6,
(iii) that the sites of phosphorylation were S e P , Serz36, SerZ4O,
Ser244, and Se?, and (iv) that these sites appeared to be
phosphorylated in the order Ser236 + Ser2= + SeS40 + Ser2@
+ Ser247 (12). This last finding was consistent with earlier
results observed in vivo (13, 14). Preliminary studies with
phosphorylated S6 derived from mitogen-stimulated Swiss
3T3 cells indicate the same sites of phosphorylation (32).
Thus, in vivo phosphorylation of S6 is a highly controlled
response which, in contrast to recent claims (41), appears to
be limited to a unique set of phosphorylation sites.
In the first studies describing an activated S6 kinase from
mitogen-stimulated cells, we showed
activity from cell extracts required the presence of &glycerol
phosphate and EGTA (15). Contrary to @-glycerol phosphate,
which was later shown to prevent dephosphorylation and
inactivation of the kinase (16, 17), the effect of EGTA was
reversible (15). From the results presented in Table I, it would
appear that EGTA chelates heavy metals in the extract, which
at low concentrations inhibit the activity of the 70K S6 kinase.
The inhibitory action of monovalent salt (Table I) is more
difficult to understand. This is a troublesome finding, since
in the absence of salt, ribosomes begin to unfold and aggregate
(46, 47). Obviously, such a structural change may alter the
pattern of phosphorylation in vitro, a possible explanation for
no detectable phosphorylation of SeP7 (see below).
Examination of the primary sequence surrounding the S6
phosphorylation sites does not reveal why one serine rather
than another is a preferred substrate (12). To better under-
stand this problem as well as the reason why Ser247 is such a
poor substrate, we first attempted to model the tertiary struc-
ture for the carboxyl terminus of S6, beginning with Lyssa.
One of the possibilities was to fold the peptide into an a-helix
and then to minimize this conformation to the lowest energy
state (48). Under these conditions the a-helix was stable
through GluZ4', but the last four amino acids, beginning with
that recovery of full
In Vitro S6 Phosphorylation Sites
Ser248, did not remain in the
hydrogen bonds (Fig. 7). It is possible, however, that these
last four amino acids are instead held in the helix by inter-
action with other ribosomal proteins or 18 S rRNA. Although
this structure is strictly hypothetical, support
pattern comes from examination of the x-ray crystal struc-
tures of proteins such as subtilisin (49), which in part contains
Closer examination of the a-helix (Fig. 7) reveals that if it
were to continue through to the end
Ser244" , S ~ I - ' ~ ~ , and Ser247 would all lie on the same side of the
helix because of the lack of
for this folding
of the peptide then Ser2:"j,
helix with their hydroxyl groups facing outward in the same
direction. Although Ser':'s is not on the same
its hydroxyl group faces in the same direction. In contrast,
Ser'42 and Ser'4fi are on the opposite side of the helix. Thus it
is possible to envision a progressive model of phosphorylation
in which the kinase begins by phosphorylating S e P and
Ser':'' and then moves along the helix phosphorylating SerL4",
Ser'44, and Ser247. The order of phosphorylation is also con-
sistent with the presence of the 3 arginines at the amino
terminus of the peptide, which create a positively charged
environment around the first 2 serines. The electropositive
amino terminus of the peptide and the permanent
generated by the helix introduce strong coulombic interac-
tions, which make the last site
much less favorable than the earlier sites and
explain some of the in vitro and in vivo results discussed
above. Though this model is consistent with much
known data, its validity remains to be tested. This
difficult in a dynamic multiprotein-RNA complex such as the
ribosome. However, we have recently found that a synthetic
peptide based on the sequence above appears to be as good a
substrate for the 70K S6 kinase as S6 within the ribosome.3
The tertiary structure of this peptide could in future be
examined by physical chemical techniques such as NMR. The
resulting information would be useful in
mechanism of S6 phosphorylation as well as its role in regu-
lating protein synthesis.
side of the helix,
of phosphorylation, Ser'47,
Acknowledgments-We would like to thank Michel Siegmann and
Renate Matthies for their technical assistance during these studies
and Drs. Brian Hemmings and Kathryn Edson
reading of the manuscript. We are also indebted to Carol Wiedmer
and Margaret Treherne for typing the manuscript.
for their critical
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In Vitro S6 Phosphorylation Sites
The concentraclon of M g ' .
the StOlChlometrlC Mg"
Indrcatlng that Mg"
klnase. Furthermore. ln the presence of 5 mM Mg", most CatlonS tested proved
to be lnhibitory in the low pmolar range; the exceptions Were CaZt. Llf and Na'
requlred to Support opflmum S6 phosphorylat.lon exceeds
ConcentratLon needed to form the ATP-Mg" complex.
also srimulates the reactmn by lnteractlng wlth the
phosphorylate 56 ~n vltro rapldly decreases durlng the reactLon (151. Thls
effect cannot be attributed to an inhibitor I" the cell extract
results are obtained wlth the pvritled Cyclohexlmide-activated 7bK 56 kmase
IFlg. 21. or LO lnhlbltlon by the substrate. as the concentraLlOn of 40s
rlbosomal Subunits 1s at least one-tenth that of the K .
and ATP the rate at which mltOgen-aCfIvaced IT3 cell extracts
Mechanism of AutoDhoSDholYlation - W e mitlally showed that rn the presence
as s m i l a r
IH. Flotow and G.T..
It 1s known that autophosphorylation can have a dramatic effect on the activity
of a kmase towards Its SubSrrate 136-381.
reaction 1 . 2 moles of phosphate are mcorpordted into the kinase per male Of
Durlng the S6 phosphorylstmn
Effect Of different caflons on s6 klnase act~vlfy.
The ICsl was determined at 5 mM Mg".
7 0 pM
In Vitro S6 Phosphorylation Sites Download full-text
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