(Received for publication, October 18, 1994; and in revised form, November 18, 1994)
From the
The role of the p90 ribosomal protein S6
kinase/mitogen-activated protein kinase (RSK/MAPK) signaling pathway in
regulating glycogen synthase kinase-3 (GSK-3) activity was
investigated. In vitro studies showed that GSK-3 was
inactivated by 50% upon incubation with RSK purified from epidermal
growth factor (EGF)-stimulated NIH/3T3 cells. Subsequently, the effect
of EGF on GSK-3 activity was measured in NIH/3T3 cells that stably
overexpressed mutated forms of MAPK kinase (MAPKK). The activation of
RSK by EGF was markedly decreased in cell lines expressing the dominant
negative MAPKK mutants S222A and K97A and was increased in cells
expressing the S222E mutant as compared with control cell lines. EGF
induced a rapid decrease in GSK-3 activity (50%) in control and
S222E cells; however, only 25 and 10% inhibition in GSK-3
activity
was observed in cell lines expressing the dominant negative mutants
K97A and S222A, respectively, suggesting that inhibition of GSK-3 was
partially blocked in these cells. Taken together, these results suggest
that the action of EGF on GSK-3 inactivation is mediated by the
RSK/MAPK signaling pathway in NIH/3T3 cells and provide evidence for a
mechanism regulating GSK-3 activity in intact cells.
Glycogen synthase kinase-3 (GSK-3) ()was originally
discovered as a protein kinase that phosphorylates and inactivates
glycogen synthase (2) and was found to be identical to factor
A that activates the MgATP-dependent form of protein
phosphatase-1(3, 4) . GSK-3 was further identified as
a multisubstrate protein kinase acting on many substrates such as ATP
citrate lyase(5) , the regulatory subunit of cyclic
AMP-dependent protein kinase (RII)(6) , transcription factors
such as c-jun, c-myc, c-myb, and
CREB(7, 8, 9, 10) , the eukaryotic
initiation factor (eIF-2B)(11) , and the microtubule-associated
protein, tau(12) . Molecular cloning from a rat brain library
revealed two GSK-3 isoforms termed
and
, which are 98%
identical in their kinase domains(13) . Mammalian GSK-3
showed close homology to the Drosophilazesta-white
3/shaggy gene product involved in fruit fly
development(14, 15) . In the budding yeast Saccharomyces cerevisiae the MCK1 and MDS1 gene products,
playing an important role in the chromosome segregation process, also
showed high homology with shaggy and GSK-3(16) . Taken
together, these data suggest that GSK-3 may play an important role in
cell proliferation, differentiation, and development.
Little is
known about the regulation of GSK-3 by growth factors and hormones.
Initially it was shown that insulin rapidly decreased the activity of a
protein kinase in rat adipocytes, which was subsequently identified as
GSK-3(17, 18) . It is currently accepted that GSK-3
activity is regulated by phosphorylation. Protein kinase C was reported
to phosphorylate GSK-3 but not GSK-3
in vitro,
decreasing its activity by 50%(19) . The ribosomal protein S6
kinase, RSK
, a major substrate of MAPK, was also shown
to directly phosphorylate and inactivate both GSK-3 isoforms in
vitro(20, 21) . More recently, GSK-3 was shown to
be activated by tyrosine phosphorylation(22, 23) ; the
phosphotyrosine site was identified as tyrosine 216(22) , a
position analogous with the TEY motif found in MAPKs. Like MAPK,
GSK-3
appears to be a dual specific enzyme, but unlike MAPK, it is
differentially regulated by tyrosine and serine/threonine
phosphorylation(24) . Still, a key question is how the enzyme
is regulated in vivo.
In these studies we have examined the
regulation of GSK-3 activity by EGF in NIH/3T3 cell lines that
overexpress mutated forms of MAPK kinase (MAPKK)(1) . These
MAPKK mutants included mutations at serine 222, one of the sites
essential for enzyme activity(25, 26) , to glutamic
acid or alanine (S222E and S222A, respectively), and a mutation at
lysine 97 (K97A). We report that EGF induced a rapid decrease in
GSK-3 activity in control and S222E mutant cells. However, in cell
lines expressing the dominant negative mutants S222A and K97A, which
showed inhibition of RSK activation by EGF, the decrease in GSK-3
activity was partially blocked. Our studies suggest that EGF-induced
GSK-3 inactivation is regulated by the RSK/MAPK signaling pathway.
Figure 1:
Effect of RSK on GSK-3-catalyzed
phosphorylation of P-GS1. The P-GS1 peptide (final concentration, 60
µM) was incubated with purified GSK-3 (5 µl of 8
units/ml) together with Mg-[-
P]ATP and RSK (opencircles) that had been immunoprecipitated from
EGF-stimulated NIH/3T3 cell lysates or from lysates treated with
preimmune serum (closedcircles). Phosphorylation of
the peptide was determined by counting at the indicated times. Results
are presented as the amount of phosphate incorporated into the P-GS1
peptide present in the reaction mixture.
Figure 2: Activation of RSK in EGF-stimulated MAPKK-transfected cell lines. MAPKK-transfected cells were treated with EGF (30 ng/ml) for the indicated times. RSK activity was assayed by the immunocomplex protein kinase assay using S6 peptide as the substrate. Results are the means of duplicates from one representative experiment. Opencircles, control cells; closedcircles, S222E cells; opensquares, K97A cells; closedsquares, S222A cells.
The fact that RSK was
differentially activated in the MAPKK-transfected cell lines (Fig. 2) prompted us to examine the effect of EGF on GSK-3
activity in these cells. The presence of GSK-3 in these cells was
detected by Western blot analyses performed on cell lysates that were
immunoprecipitated with specific antibodies against
GSK-3(28) . A single band of 45 kDa, equally distributed
in all cell lines, was detected (Fig. 3) and indicated that
these antibodies specifically immunoprecipitated GSK-3
; such
precipitates have been shown to serve as a reliable tool for measuring
GSK-3 activity in crude cell preparations(23, 28) ,
and this technique was then applied here. Transfected cell lines were
treated with EGF (30 ng/ml) for different periods of times. GSK-3
was immunoprecipitated from cell lysates, and their GSK-3 kinase
activity was measured using P-GS1 peptide as the substrate. As shown in Fig. 4A, EGF caused a rapid decrease in GSK-3
activity (50% decrease after 4-5 min) in both control and S222E
cell lines. However, a decrease of only 10 and 25% in GSK-3
activity was observed in S222A and K97A cells, respectively (Fig. 4A), indicating that inactivation of GSK-3
was impaired in these cells. In addition, the recovery of GSK-3
activity was quicker and more complete in S222A and K97A cells than in
control or S222E cell lines. GSK-3
activity was also assayed using
inhibitor-2 (I2) as the substrate (Fig. 4B). Consistent
with the previous results, phosphorylation of I2 by GSK-3
was
markedly decreased in control and S222E cell lines treated with EGF (Fig. 4B, lanes1-4). Little or
no effect on I2 phosphorylation was detected in S222A and K97A cells (Fig. 4, lanes 5-8). (In this particular
experiment, the basal activity of GSK-3
in S222A cells was
somewhat low relative to the other cell lines (Fig. 4B, lanes7 and 8). Generally, however, the
basal levels of GSK-3
activity were comparable in all cell lines.)
Although higher levels of RSK activity were detected in S222E cells (Fig. 2), EGF-induced GSK-3 inactivation was found to be
comparable with that of the control cells. We cannot provide a complete
explanation for this observation, but it is possible that in a complex
cascade such as that operating here, a 1:1 relationship between the
activity of upstream and downstream kinases may not always exist.
Figure 3:
Expression of GSK-3 in
MAPKK-transfected cell lines. Cell lysates, normalized for the amount
of protein, were incubated with specific antibodies against GSK-3
as described under ``Materials and Methods.'' Washed
immunoprecipitates were subjected to 10% SDS-gel electrophoresis,
blotted onto polyvinylidene difluoride membranes incubated with
anti-GSK-3
antibodies, and detected with horseradish
peroxidase-labeled protein A. Lane1, control cells; lane2, S222E cells; lane3, K97A cells; lane4, S222A
cells.
Figure 4:
Inactivation of GSK-3 in
EGF-stimulated MAPKK-transfected cell lines. NIH/3T3 cells were treated
with EGF for the indicated times. GSK-3
was immunoprecipitated
from cell lysates as described under ``Materials and
Methods.'' A, GSK-3 activity was determined in the
immunoprecipitates using P-GS1 peptide as the substrate and is given as
the percentage of enzyme activity measured in non-stimulated cells.
Results are the mean of five independent experiments; S.D. was less
than 10%. Opencircles, control cells; closedcircles, S222E cells; opensquares,
K97A cells; closedsquares S222A cells. B,
cells were treated with EGF for 6 min, and GSK-3
was
immunoprecipitated from cell lysates as described under
``Materials and Methods.'' I2 (10 µg) was added to the
immunoprecipitates, and its phosphorylation was analyzed as described
under ``Materials and Methods.'' Lanes 1 and 2, control cells; lanes3 and 4,
S222E cells; lanes5 and 6, K97A cells; lanes7 and 8, S222A
cells.
The fact that GSK-3 is constitutively active in resting cells (22, 23) is consistent with the concept that this
enzyme might be negatively regulated by growth factors. Little is
known, however, about the mechanism that might be involved in such
regulation. Nor was it known whether growth factors or hormones other
than insulin could cause GSK-3 inactivation. Here we show that EGF
causes a rapid decrease in GSK-3 activity in NIH/3T3 cells and
that EGF-induced GSK-3
inactivation is partially prevented in
cells expressing the dominant negative mutants (S222A and K97A) of
MAPKK. Since the activation of RSK, MAPK, and MAPKK by EGF was markedly
suppressed in these cells (1) (Fig. 2), these results
suggest that EGF action on GSK-3 inactivation is mediated by the MAPK
signaling pathway. These data alone cannot be used, however, to
determine which kinase (i.e. MAPKK, MAPK, RSK, or an as yet
unknown downstream kinase) is directly involved in GSK-3 inactivation,
but in vitro studies suggest that RSK is the component
inactivating GSK-3 (20, 21) (Fig. 1). These
data do not exclude the possibility that other mechanisms might also be
involved in the regulation of GSK-3. For example, as noted earlier,
tyrosine phosphorylation has also been shown to regulate the
enzyme(22, 24) , suggesting that activation of
tyrosine phosphatase could play a role in GSK-3 inactivation. Indeed, a
decrease in phosphotyrosine content was reported to cause inactivation
of the enzyme in A431 cells(23) .
The question as to whether the Ras/MAPK signaling pathway mediates the action of insulin and related factors such as EGF (also see (32) ) on glycogen metabolism is still not clear. Studies in rat adipocytes suggested that the MAPK signaling pathway is not sufficient for mediating the action of insulin on glycogen synthase and glucose transport(29, 30) . In contrast, expression of activated mutants of Ras in 3T3-L1 adipocytes was shown to mimic the effect of insulin on membrane trafficking of glucose transporters(31) . If it is assumed that GSK-3 does play an important role in regulating glycogen synthase activity, our studies could imply that the RSK/MAPK signaling pathway mediates the action of hormone or growth factor regulation of the enzyme. However, it is quite possible that inactivation of GSK-3 through the MAPK cascade is not sufficient by itself to induce these changes. Additional studies are needed to clarify these points.
Another laboratory has reported similar results to those presented here; this came to our attention during the review process. These studies indicate that insulin-like growth factor and EGF induce inactivation of GSK-3 in intact cells and show that the MAPK cascade appears to play a role in regulating GSK-3 activity(33, 34, 35) . This conclusion is drawn on the basis of utilizing wortmannin to block MAPK activation and its effect on GSK-3.