(Received for publication, February 27, 1995; and in revised form, May 23, 1995)
From the
A unique and highly conserved structural feature of 90-kDa
ribosomal S6 kinase (p90
or RSK) is the presence
of two non-identical kinase domains. To explore the mechanism of RSK
activation, a cloned human RSK cDNA (RSK3) was used to generate and
characterize several site-directed RSK mutants: K91A (N-Lys,
NH
-terminal ATP-binding mutant), K444A (C-Lys,
COOH-terminal ATP-binding mutant), N/C-Lys (double ATP-binding mutant),
T570A (C-Thr, mutant of the putative MAPK phosphorylation site in
subdomain VIII of the C-domain), S218A (N-Ser, mutant of the
corresponding NH
-terminal residue). Epitope-tagged RSKs
were expressed in transfected COS cells followed by immunoprecipitation
with or without prior in vivo epidermal growth factor
stimulation. Kinase activity (S6 peptide) of N/C-Lys and N-Lys was
ablated (and partially impaired with N-Ser). In contrast, both C-Lys
and C-Thr retained high levels of kinase activity and were capable of
responding to stimulation. C-Lys also retained partial kinase activity
toward other substrates (c-Fos, S40 ribosomes, protein phosphatase 1
G-subunit, histones, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide))
whereas NLys did not. The isolated NH
- and COOH-terminal
domains were also expressed; the C-domain was inactive, whereas the
N-domain retained partial activity. Relative to wild-type, both N-Lys
and C-Lys (as well as N-Ser and C-Thr) underwent partial in vitro autophosphorylation that was further stimulated by EGF protein
tyrosine phosphatase. We conclude that 1) the NH
-terminal
RSK kinase domain mediates substrate phosphorylation; 2) both domains
contribute to autophosphorylation; 3) the putative MAPK phosphorylation
site is not required for growth factor-stimulated autophosphorylation
or kinase activation.
Protein serine/threonine kinases mediate a broad array of cellular functions including the stimulation of proliferation and differentiation by cytokines and growth factors(1, 2, 3) , cellular oncogenesis(4) , regulation of the cytoskeleton(5) , responses to stress (e.g. changes in osmolarity, UV light, ionizing radiation, 6-9), the regulation of metabolism by insulin and other hormones(10, 11, 12) , and possibly apoptosis(13) .
The p90 ribosomal S6 kinase (p90 or RSK) (
)has been implicated as an important
participant in several of these critical cellular events. Thus,
activation of RSK coincides with oncogenic transformation(14) ,
stimulation of G
/G
transition(1, 15, 16) , T cell
activation(17) , stimulated differentiation of PC12
cells(18) , platelet activation(19) , and cellular
responses to heat shock (20) or ionizing radiation(9) .
RSK has also been demonstrated within the cell nucleus and is thought
to phosphorylate nuclear target substrates (21, 22, 23) . An additional important role
for RSK involves its phosphorylation of the G-subunit of protein
phosphatase 1 and glycogen synthase kinase-3, since these events
contribute to the regulation of glycogen metabolism by
insulin(10, 24, 25, 26, 27, 28) .
Molecular cloning of a RSK cDNA from Xenopus ovarian tissue
revealed a predicted protein with a strikingly different structure from
other Ser/Thr kinases(29) . Thus, the RSK polypeptide contained
two non-identical complete kinase domains. The NH-terminal
kinase domain is related to protein kinases C and the catalytic subunit
of cAMP- and cGMP-dependent kinases (40-45%); whereas the
COOH-terminal domain bears 30-35% homology to phosphorylase b kinase and calcium/calmodulin kinases(29, 30) .
Molecular cloning of RSK isoforms from the chicken and
mouse(31) , rat(32) , and human (33, 34) have demonstrated precise conservation of
this unique feature along with strong overall homology (75-85%)
between different 724-752 amino acid isoforms and between
species. Strong evolutionary pressure to conserve this structure was
further demonstrated by the report of a highly related Drosophila RSK homolog(35) . In spite of intensive recent interest in
RSK, the functional importance of dual ATP-binding sites and distinct
kinase domains remains obscure. Given that RSK exhibits broader
substrate specificity than the other class of S6 kinase,
p70/85
(12, 36) , it is possible
that individual RSK kinase domains mediate phosphorylation of
different substrates or have other roles in substrate recognition or
activation.
RSK is regulated by phosphorylation on Ser (mostly) and
Thr residues which is mediated by upstream Ser/Thr kinases and by
additional autophosphorylation(12, 32) . Furthermore,
the enzyme does not contain phosphotyrosine and can be dephosphorylated
and deactivated by protein phosphatase
2A(12, 25, 32, 37) . Several lines
of evidence suggest that isoforms of mitogen-activated protein kinase
(MAPK), in particular p44 (ERK1) and p42
(ERK2), are upstream activators of
RSK(25, 32, 37, 38, 39, 40) .
Thus, in vitro phosphorylation of RSK by ERK1,2 results in
enzyme activation(25, 32, 37) . In addition,
ERK1,2 MAPKs are detected in fractions from mitogen-stimulated cell
extracts which mediate RSK
phosphorylation(38, 41, 42, 43) . By
sequencing of a chymotryptic phosphopeptide after in vitro phosphorylation of a RSK isoform (RSK2) by MAPK, Sutherland et
al.(43) identified two predominant Thr phosphorylation
sites within the sequence TPCYTA located upstream of the APE motif in
subdomain VIII of the second kinase domain. These residues are
conserved in all known RSK
isoforms(25, 31, 33) . Since the first Thr
residue is followed by Pro, they suggested that this was the major site
of transphosphorylation by MAPKs which are known to be
proline-directed(5, 43) . Furthermore, MAPK
phosphorylated only the first Thr in a peptide containing both
residues(43) .
In order to explore the mechanism of RSK
activation and the functions of the two-domain structure, we expressed
epitope-tagged wild-type and mutated versions of a cloned human RSK
cDNA (44) in COS cells and characterized their function. Here
we report: (i) phosphorylation of several known RSK substrates is
mediated exclusively by the NH-terminal kinase domain, (ii) in vitro RSK autophosphorylation is partially impaired by
mutation of either ATP-binding site, (iii) mutation of the putative
MAPK-phosphorylated Thr is associated with normal kinase activity and a
preserved ability to respond to growth factor stimulation, (iv) an
NH
-terminal Ser residue that is important for maximal
kinase activity is identified.
Two additional
constructs designed for expression of the isolated
NH-terminal (RSK
C) and COOH-terminal kinase
(RSK
N) domains were prepared as follows: for RSK
C the
wild-type RSK expression vector (pMT2-HA-RSK3) was digested with BsgI and DraIII followed by religation using a short
double-stranded oligonucleotide adapter. This resulted in the in-frame
removal of the entire COOH-terminal kinase domain via deletion of
residues 384-689. For RSK
N, a XhoI-MluI
fragment encoding residues 33-219 was removed from
pMT2-HA-RSK3/N-Ser (which had been previously modified to include a
unique MluI site), followed by in-frame blunt-ended
religation.
For protein tyrosine phosphatase
treatment, aliquots of immunoprecipitated proteins were incubated in 15
µl of 30 mM HEPES, 150 mM NaCl, 50 mM -glycerophosphate, and 5 mM dithiothreitol, with or
without 0.8 milliunits protein tyrosine phosphatase/ml for 20 min at 30
°C. Reactions were terminated by washing once with 1 ml of ice-cold
KA buffer and resuspending in ice-cold KA buffer
followed by determination of kinase activity and autophosphorylation as
described above.
To investigate the role of the two kinase domains of the 90-kDa RSK in substrate phosphorylation and in growth factor-mediated activation of the enzyme, oligonucleotide-directed mutants of a human RSK cDNA were generated and characterized. For this purpose, we used the cDNA encoding a 733-amino-acid isoform designated as RSK3(44) . RSK3 is homologous (75-84% overall identity) to the two other known mammalian RSK isoforms (31, 33, 34) with a high degree (86-92%) of amino acid identity between the corresponding kinase domains in each isoform.
In each of the two kinase domains, the
invariant lysine (Lys and Lys
) in subdomain
II (30) was mutated to alanine to create N-Lys and C-Lys
ATP-binding site mutants (Fig.1A). Based on earlier
studies(30, 49) , each of these two mutations would be
expected to markedly impair phosphotransferase activity mediated by the
respective kinase domain. Also, a double kinase mutant (N/C-Lys) where both lysine residues were changed to alanine
was created. Furthermore, to specifically address the possible role of
Thr
in subdomain VIII of the COOH-terminal domain in
growth factor-mediated kinase activation, this residue was also mutated (C-Thr, Fig.1A). This residue in RSK2 (also
known as ISPK-1 or MAPKAP-1) Thr
(34) has been
implicated as the predominant site of MAPK (ERK2)
phosphorylation(43) . The corresponding residue in the
NH
-terminal kinase domain, Ser
, was also
mutated (N-Ser, Fig.1A). Both Ser
and Thr
are located within 20 residues upstream of
the highly conserved A(S)PE motif present in all protein kinases, a
region known to often contain phosphorylation sites that are critical
for catalytic function(30) .
Figure 1:
Construction and
expression of site-directed RSK mutants. A, schematic drawing
of a 733-amino acid human RSK polypeptide (RSK3). Conserved regions
within protein kinase subdomains I-II and VIII, according to Hanks et al.(30) , are shown as filled and hatchedboxes, respectively. Four individual residues
which were changed into alanine (Lys, Ser
,
Lys
, and Thr
) are shown in bold.
The five mutant RSK polypeptides that were generated by site-directed
mutagenesis are designated as follows: N-Lys (K91A), N-Ser (S218A),
C-Lys (K444A), C-Thr (T570A), and N/C-Lys (K91A and K444A double
mutant), where N and C denote the amino- and carboxyl-terminal kinase
domains, respectively. B, immunoblotting of immunoprecipitated
HA-tagged mutant and wild-type RSK proteins. In this example,
HA-epitope-tagged cDNAs encoding wild-type (WT) or mutant RSK
polypeptides or vector alone (Vect.) were transfected into COS
cells. Cell lysates were immunoprecipitated with 12CA5 antibody. Equal
aliquots of 12CA5 immunoprecipitates were analyzed by SDS-PAGE followed
by immunoblotting with anti-RSK(N67) antibody. A single 85 kDa protein
band of expected size and similar intensity was detected in each case.
Densitometry was performed using autoradiograms of immunoblots from 9
experiments. Mean (± S.E.) protein levels (as percent of
wild-type level) for each of the mutants were as follows: 120 ±
34 (N-Lys), 115 ± 27 (N-Ser), 85 ± 19 (C-Lys), 121
± 23 (C-Thr), 117 ± 33
(N/C-Lys).
After using in vitro transcription/translation to verify that these five mutant and
wild-type RSK polypeptides were synthesized with equal efficiency (not
shown), the cDNA coding regions were cloned into pMT2-HA(32) ,
an expression vector containing the 9-residue influenza virus HA
epitope tag. This allowed for the recombinant proteins to be isolated
by immunoprecipitation with a monoclonal antibody (12CA5) directed
against the HA epitope after transient expression in transfected COS-7
cells. In each of several experiments that were performed, we verified
that similar amounts of wild-type or mutated RSK proteins were present
in 12CA5 immunoprecipitates by immunoblotting with a polyclonal
antibody raised against the NH terminus of the human RSK3
protein (Fig.1B). Analysis of immunoblotting data from
nine independent experiments showed that mean levels of
immunoprecipitated RSK protein for each of the five mutants did not
differ from wild-type (see legend to Fig.1B).
We
first compared the activities of HA-tagged mutant and wild-type
proteins using an in vitro immune-complex kinase assay with
immunoprecipitates from transfected cells. The cells were serum starved
or serum starved followed by stimulation with EGF which has been
reported to activate RSK(16, 28) . A peptide
(RRRLSSLRA) corresponding to amino acids 231-239 of ribosomal S6
protein was tested since it is known to be avidly phosphorylated by
RSK(12) . For each experiment, a vector-only transfected
control was included. Levels of background substrate phosphorylation
were consistently less than 1% of kinase activity achieved using
wild-type RSK transfected cells. As shown in Fig.2, wild-type
RSK activity increased by 3.5-fold after in vivo stimulation.
As predicted, the double ATP-binding site mutant was devoid of kinase
activity. Of the two single ATP-binding site mutants, the N-Lys mutant
showed no measurable activity, while the C-Lys mutant retained
substantial kinase activity (approximately 50% of wild-type both in the
serum-deprived and stimulated conditions). Thus, EGF stimulation of
C-Lys kinase activity was also preserved. Examination of the C-Thr
mutant revealed that it also retained high levels of kinase function
and was still capable of responding to growth factor stimulation
(2.4-fold). Mutation of the corresponding NH-terminal Ser
residue resulted in modest impairment of kinase activity from both
serum-deprived and stimulated cells (
90% reduced).
Figure 2:
Kinase activity of mutant and wild-type
RSK polypeptides derived from serum-deprived or EGF-stimulated COS
cells. Following transfection of COS-7 cells with epitope-tagged mutant
or wild-type RSK cDNAs, cells were serum-starved for 6 h and then
stimulated (hatched bars) or not (filled bars) with
100 ng/ml EGF for 15 min. Cell lysates were immunoprecipitated with
HA-epitope antibody 12CA5. Equal aliquots of immunoprecipitated protein
were assayed for protein kinase activity using S6 peptide (RRRLSSLRA)
as a substrate. Separate aliquots of 12CA5 immunoprecipitates were
analyzed by immunoblotting (Fig.1B) to verify that
similar amounts of RSK proteins were present. The results are expressed
as a percentage of EGF-stimulated kinase activity achieved using
wild-type (WT) RSK protein. Data are means ± S.E. of
eight experiments; mean levels of stimulated WT RSK kinase activity
were 2.8 ± 0.5 pmol of ATP transferred/min/reaction (6.2
10
counts/min/10 min reaction); background enzyme activity
obtained with immunoprecipitates from vector-only transfected cells was
consistently <1.0%. Similar results were obtained in a more limited
number of experiments where cells were stimulated with tetradecanoyl
phorbol acetate.
Although the
N-Lys mutant was unable to phosphorylate S6 peptide, we sought to test
whether the two kinase domains might each phosphorylate different
substrates. Therefore, we isolated N-Lys, C-Lys, and wild-type RSK from
stimulated cells and compared their ability to phosphorylate eight
different polypeptides that were previously shown to be in vitro substrates for
RSK(15, 21, 25, 32, 44, 50) .
As shown in Table1, kinase activity of the N-Lys mutant was very
low or unmeasurable. In contrast, the C-Lys mutant retained kinase
activity toward each of the eight tested substrates. Interestingly, in
all cases C-Lys kinase activity was diminished relative to wild-type
RSK. This, suggests that both kinase domains may be required for
maximal activity toward various substrates. Although it remains
possible that the COOH-terminal kinase domain might be able to trans-phosphorylate (unknown) in vivo RSK
substrate(s), these data strongly suggest that substrate
phosphorylation is mediated exclusively by the NH-terminal
kinase domain. An additional, but less likely, possibility is that
activation of the COOH-terminal kinase domain requires a unique
upstream element or pathway which was not stimulated by EGF, serum, or
tetradecanoyl phorbol acetate or was absent from COS cells.
In order
to assess whether either RSK kinase domain was capable of functioning
independently, we generated truncated proteins where either the
COOH-terminal (RSKC) or NH
-terminal (RSK
N) domain
was deleted. After immunoprecipitation of lysates derived from
transfected cells, epitope-tagged RSK
C or RSK
N proteins were
readily detected by immunoblotting as 53- or 60-kDa proteins,
respectively (not shown). In subsequent immune-complex kinase assays,
we found that the isolated NH
-terminal domain (RSK
C)
retained low level kinase activity toward S6 peptide (5-6%
relative to wild-type RSK). In contrast, the RSK
N mutant displayed
no detectable kinase activity (above background values obtained with
vector-only transfected cells). Since the full-length molecule with
mutated COOH-terminal Lys was apparently more active than the isolated
NH
-terminal domain, this suggests that the presence of the
COOH-terminal domain, even when inactive, acts to facilitate the
function of the NH
-terminal domain.
Available evidence
suggests that RSK undergoes autophosphorylation coincident with kinase
activation(12, 32) . To further address the potential
functional role of each domain, and in particular to assess whether the
COOH-terminal kinase contributes to autophosphorylation, we performed in vitro RSK autokinase assays with or without prior in
vivo stimulation with EGF. As shown in Fig.3A,
incubation of wild-type RSK with [-
P]ATP
resulted in substantial in vitro phosphorylation that was
further augmented (3.5-fold) by EGF stimulation. We were surprised to
find a small amount (
10% compared to wild-type) of RSK
phosphorylation in the case of the double ATP-binding mutant (N/C-Lys, Fig.3A). This result suggested
contamination with a coimmunoprecipitated kinase. Since MAPK and RSK
have been reported to be
coimmunoprecipitated(50, 51) , we tested the effect of
pretreatment with protein tyrosine phosphatase (PTPase) which is known
to fully inactivate MAPK. Using this approach, we found that prior
PTPase treatment abrogated phosphorylation of N/C-Lys, whereas
wild-type RSK phosphorylation was reduced by approximately 25% (Fig.3A). Importantly, PTPase treatment did not affect
the subsequent determination of kinase activity toward S6 peptide (not
shown). The fact that both N-Lys and N/C-Lys mutants lacked detectable
kinase activity toward S6 peptide (Fig.2) also argues strongly
that potential coimmunoprecipitated kinase(s) did not contribute to the
enzyme activities that were measured. In order to eliminate the effects
of the coimmunoprecipitated MAPK-like activity on in vitro RSK
autophosphorylation, subsequent experiments were conducted after
pretreatment with PTPase.
Figure 3:
In vitro RSK autophosphorylation. A, RSK phosphorylation and effect of PTPase treatment. In this
example, COS cells transfected with the double-lysine mutant (N/C-Lys), wild-type RSK (WT), or the expression
vector alone (Vect.) were serum starved(-) or starved
followed by EGF stimulation (+). Cell lysates were
immunoprecipitated with antibody 12CA5 followed by in vitro treatment with (+) or without(-) PTPase and subsequent
incubation with [P]ATP in the absence of
substrate as described under ``Materials and Methods.''
Proteins were resolved by SDS-PAGE followed by autoradiography.
Phosphorylated 85 kDa bands corresponding to RSK3 proteins are
depicted. B, phosphorylation of wild-type (WT) and
mutant RSK proteins. COS cells transfected with wild-type or mutant RSK
cDNAs were serum-starved followed by stimulation with (hatched
bars) or without (filled bars) EGF. Immunoprecipitated
RSK proteins were pretreated with PTPase followed by determination of in vitro autophosphorylation as described above. Radiolabeled
RSK proteins were quantitated by PhosphorImager analysis. Results are
expressed as a percentage of EGF-stimulated wild-type RSK
phosphorylation. Data are means ± S.E. of five independent
experiments.
As shown in Fig.3B, both
serum-deprived and stimulated values of in vitro RSK
autophosphorylation were modestly reduced with the C-Lys mutant. This
result supports the hypothesis that RSK autophosphorylation (and
subsequent NH-terminal kinase activation) could be
mediated, at least in part, by the COOH-terminal domain. Furthermore,
mutation of Thr
resulted in modest effects on
autophosphorylation with protein derived from both serum-deprived and
-stimulated cells, exactly mirroring the effect of this mutation on
kinase activity ( Fig.2and Fig. 3B). It
therefore appears that Thr
is not required for activation
by EGF of either autophosphorylation or kinase activity toward
substrates. The fact that the C-Thr mutant retained the ability to
respond to stimulation suggests that MAPK(s) might phosphorylate
alternative sites (on either the NH
- or COOH-terminal
domains). Alternatively, the in vivo regulation of RSKs may
involve additional upstream kinases, apart from ERK1/2 as was suggested
by others(1) . Furthermore, it is possible that the RSK3
isoform may not be exactly regulated as RSK2 (or RSK1). Indeed, we
recently showed that although RSK3 could be phosphorylated in vitro by ERK2, its phosphotransferase activity was
unaffected(44) . In contrast, parallel in vitro incubation of ERK2 with recombinant RSK1 augmented the activity of
this isoform(44) .
Despite the absence of protein kinase
activity with mutation of the NH-terminal ATP-binding site (Fig.2), EGF stimulated phosphorylation of this mutant (N-Lys)
to levels that were 15% of the wild-type (3-fold versus protein from serum-deprived cells, Fig.3B). These
results suggest that the residual capacity for RSK autophosphorylation
in this case is likely to have been mediated by the COOH-terminal
domain, although maximal in vitro RSK autophosphorylation
appears to require that both kinase domains are intact. Additional
studies may help to define whether RSK autophosphorylation occurs via
an inter- or intramolecular mechanism and whether phosphorylation of
the NH
-terminal domain might be mediated, in part, by the
COOH-terminal kinase.
Data regarding the N-Ser mutant suggested that
Ser might be important for autophosphorylation and kinase
activity. Given that the analogous residue in the catalytic domains of
several other protein Ser/Thr kinases including MAPK (Thr in the TEY
motif, 52) and MAPK kinase (Ser
in MEK1, 54, 55) are
important sites of phosphorylation by upstream activators, this residue
could represent a site of regulated phosphorylation. Therefore, we also
generated mutants where Ser
was replaced by Asp or Glu in
an attempt to mimic a potential negative phosphate charge at this site.
Both S218E and S218D epitope-tagged mutants were present at levels
similar to wild-type RSK in immunoprecipitates derived from transfected
cells (not shown). In subsequent kinase assays, when both mutants were
isolated from serum-starved cells they exhibited activity that was
17% of wild-type levels; stimulated values were
40% compared to
wild-type RSK. Thus, neither mutant displayed features suggestive of
constitutive activation. Since all three mutants of Ser
were readily stimulated by EGF (5-7-fold), this residue is
clearly not required for growth factor-stimulated RSK activation.
However, given that values of kinase activity with the Ala substituted
mutant were
4-fold lower relative to both Asp or Glu substitutions,
Ser
may represent a site of autophosphorylation that
contributes to maximal enzyme activity.
Taken together, data
presented in this study are consistent with a model whereby maximal
growth factor-stimulated RSK activation requires the participation of
both kinase domains. The COOH-terminal domain appears to participate in
regulation of RSK autophosphorylation. The NH-terminal
domain also contributes to autophosphorylation and mediates substrate
phosphorylation. Therefore, the structure of RSK provides a unique
signal transducing paradigm where what might otherwise exist as two
distinct enzymes in a kinase cascade are combined into one
interdependent unit.