(Received for publication, August 23, 1995)
From the
In the evolutionarily distant yeasts Saccharomyces
cerevisiae and Schizosaccharomyces pombe, genetic
evidence suggests that activation of pheromone-induced
mitogen-activated protein kinase (MAPK) cascades involves the function
of the p21-activated protein kinases
(PAKs) Ste20 and Shk1, respectively. In this report, we show that
purified Ste20 and Shk1 were each capable of inducing p42
activation in cell-free extracts of Xenopus laevis oocytes, while a mammalian Ste20/Shk1-related protein kinase,
p65
(Pak1), did not induce activation of
p42
. In contrast to p42
, activation of
JNK/SAPK in Xenopus oocyte extracts was induced by both the
yeast Ste20 and Shk1 kinases, as well as by mammalian Pak1. Our results
demonstrate that MAPK cascades that are responsive to PAKs are
conserved in higher eukaryotes and suggest that distinct PAKs may
regulate distinct MAPK modules.
In eukaryotes, responses to numerous types of extracellular
signals are mediated by highly conserved mitogen-activated protein
kinase (MAPK) ()modules(1, 2, 3) .
The basic MAPK module consists of a MAPK kinase kinase (MAPKKK), a MAPK
kinase (MAPKK), and a MAPK. MAPKKKs phosphorylate and activate MAPKKs,
which phosphorylate and activate MAPKs(4) . Homologs of these
kinases appear to be both structurally and functionally conserved in
evolution (3, 5, 6) . Data from yeast genetic
studies, and more recently from studies of mammalian cells, suggest
that different types of extracellular signals activate distinct MAPK
modules. For example, in the budding yeast Saccharomyces
cerevisiae, distinct MAPK modules mediate cellular responses to
mating pheromones, osmotic shock, extracellular salt concentration, and
nutrients(3) . Evidence has been obtained demonstrating the
existence of at least four distinct MAPK modules in mammalian cells,
although it is likely that the actual number of modules is
substantially
larger(2, 7, 8, 9, 10, 11, 12) .
At present, we have acquired only a limited understanding of how distinct MAPK modules are individually regulated. The best characterized mammalian MAPK cascades are those regulated by receptor tyrosine kinases, such as the epidermal growth factor and platelet-derived growth factor receptors, which signal through Ras proteins via Grb2-related adaptor proteins and Ras guanine nucleotide exchange factors(13) . Ras proteins, in turn, directly control the activity of the MAPKKK, Raf-1(14) . Substantially less is known about the regulation of MAPK cascades controlled by other types of receptors, in particular those activated by G protein-coupled seven transmembrane domain receptors (serpentine receptors)(2, 15) . However, recent studies (16, 17, 18) have provided evidence for the existence of both Ras- and Raf-independent MAPK cascades.
In the
evolutionarily distant yeasts S. cerevisiae and Schizosaccharomyces pombe, the
p21-activated protein kinases (PAKs)
Ste20 and Shk1, respectively, have been implicated as functioning
upstream of pheromone receptor-induced MAPK
modules(19, 20) . (
)Wu et al.(21) recently showed that Ste20 immune complexes
phosphorylate the S. cerevisiae MAPKKK, Ste11, implicating
Ste20 as a potential MAPKKK kinase. PAKs structurally related to Ste20
and Shk1 have been identified in mammalian
cells(22, 23) . However, it has not yet been
determined whether mammalian PAKs regulate MAPK cascades.
In this
report, we show that purified Ste20 and Shk1 protein kinases are each
capable of inducing activation of p42 in cell-free
extracts of Xenopus laevis oocytes. In contrast,
p65
(Pak1), a mammalian PAK structurally related
to Ste20 and Shk1, did not induce p42
activation in Xenopus oocyte extracts. We show that the S. cerevisiae MAPKKK, Ste11, is capable of inducing p42
activation, and that the N-terminal regulatory domain of Ste11
partially blocked Ste20-induced activation of p42
.
Finally, we show that unlike p42
, activation of a
JNK/SAPK cascade in Xenopus oocyte extracts was induced by
both the yeast Ste20 and Shk1 kinases and by mammalian Pak1. Our data
demonstrate that MAPK cascades that are activated by PAKs are conserved
in higher eukaryotes and suggest that distinct MAPK modules may be
regulated by different types of PAKs.
Genetic experiments have suggested that the S. cerevisiae and S. pombe PAKs, Ste20 and Shk1, respectively, function
upstream of mating pheromone-induced MAPK
modules(19, 20) . To examine whether
PAK-responsive MAPK cascades are conserved in evolution, we utilized a
cell-free system for examining the activation of p42
in
extracts of X. laevis oocytes(26) . This and similar
systems have previously been used to demonstrate in vitro activation of p42
induced by purified Ras, Mos,
protein kinase A, and protein kinase
C(26, 28, 29, 30) . Using this assay
system, a direct correlation between the mobility shift induced by
these various agents and activation of the kinase activity of
p42
has been demonstrated(26) . A
GST-Ste20
fusion protein (GSte20
N), lacking amino acid
residues 1-495 of the Ste20 regulatory domain, and a
GST-Shk1
N fusion protein (GShk1
N), lacking residues
1-182 of the Shk1 regulatory domain, were expressed in yeast and
purified from cell lysates using glutathione-agarose beads (see
``Materials and Methods''). Both GSte20
N (Fig. 1A) and GShk1
N (not shown) were judged to be
catalytically active, based on the ability of each to autophosphorylate in vitro. As shown in Fig. 1B, GSte20
N
rapidly induced activation of p42
in Xenopus oocyte extracts, as measured by a decrease in the mobility of
p42
. Using less than 1% of the amount of GSte20
N
protein compared with Ras protein, we detected Ste20
N-induced
p42
activation in 30 min, while
p21
-induced p42
activation required 2 h.
GShk1
N also induced activation of p42
when added to Xenopus extracts, although not as rapidly as GSte20
N (Fig. 1B). A bacterially expressed Ste20 protein that
contained a functional regulatory domain was also capable of inducing
p42
activation in Xenopus oocyte extracts,
albeit not as effectively as either GSte20
N or GShk1
N (data
not shown).
Figure 1:
Activation of p42
induced by the S. cerevisiae Ste20 and S. pombe Shk1
protein kinases in cell-free extracts of Xenopus oocytes. A, the Ste20
N protein autophosphorylates in
vitro. GST and GST-Ste20
N (GSte20
N) proteins
were expressed in yeast and purified from cell extracts using
glutathione agarose beads as described under ``Materials and
Methods.'' Kinase assays were performed in the presence (+)
or absence (-) of 5 ng of GSte20
N attached to glutathione
agarose beads and/or 250 ng of control GST beads (to ensure that the
GST moiety was not phosphorylated by GSte20
N). Samples were
resolved by 12% SDS-PAGE, then exposed to film for autoradiography. The asterisk indicates the location of GSte20
N and the
indicates the location of GST protein. B, Ste20
N
and Shk1
N induce activation of p42
in Xenopus oocyte extracts. GST GST-Ste20
N (GSte20
N) and
GST-Shk1
N (GShk1
N) proteins were expressed in yeast
and purified from cell extracts using glutathione-agarose beads, and
H-Ras[G12V] protein was purified from bacteria as described
under ``Materials and Methods.'' MAPK assays were performed
by adding 1 µg of GST, 20 ng of GSte20
N, 100 ng of
GShk1
N, or 3 µg of H-Ras[G12V] protein to 20 µl
of lysate as described under ``Materials and Methods.'' At
the indicated times, 3-µl samples were removed, mixed with 30
µl of SDS-PAGE sample buffer, and boiled for 5 min. Samples were
resolved by 15% SDS-PAGE, transferred to nitrocellulose, and blots
probed as indicated under ``Materials and Methods.'' The arrow indicates the activated, slower migrating form of
p42
.
To determine whether p42 activation was
dependent on Ste20 catalytic function, we introduced a mutation into
the GSte20
N coding sequence at Lys
, a residue that
is conserved in protein kinases(31) .
GSte20
N[K649R] was catalytically inactive, as determined
by its inability to autophosphorylate, and did not induce
p42
activation when added to Xenopus oocyte
extracts (Fig. 2). Thus, p42
activation induced
by Ste20 requires that the protein be catalytically active.
Figure 2:
Ste20N-induced p42
activation is dependent on Ste20 catalytic function. Fusion
proteins were purified from yeast cell extracts as described under
``Materials and Methods.'' Kinase assays (left side of
panel) were performed as described in Fig. 1A using GSte20
N or GSte20
N[K649R] fusion
proteins. The ability of GSte20
N or GSte20
N[K649R]
fusion proteins to induce p42
activation in Xenopus oocyte extracts (right side of panel) was measured as
described in Fig. 1B.
Genetic
data suggest that the Ste11 protein kinase functions downstream of
Ste20 in the S. cerevisiae pheromone response pathway (19) . Therefore, we tested whether the N-terminal regulatory
domain of Ste11 could inhibit activation of p42 induced
by GSte20
N in Xenopus oocyte extracts. The Ste11
regulatory domain (residues 1-415) was purified from a bacterial
expression system as a GST-fusion protein. As shown in Fig. 3A, GSte11
C partially blocked p42
activation induced by GSte20
N in Xenopus oocyte
extracts. This result is consistent with two possible interpretations:
(i) the Ste11 N terminus inhibits the activity of a protein activated
by Ste20 in Xenopus oocyte extracts or (ii) Ste11 is a
substrate of Ste20.
Figure 3:
Effect of S. cerevisiae Ste11 and
Ste11C proteins on p42
activation. A, Xenopus oocyte extracts were incubated with 2 ng of
GSte20
N glutathione-agarose beads and either 32 ng of GSte11
C
glutathione-agarose beads or 8 µl of glutathione-agarose beads
alone. p42
activation was measured as described in the
legend to Fig. 1and is expressed as the percentage of
p42
in activated form (p42
activated/total p42
) as determined by
densitometry. B, full-length GST-Ste11 fusion protein was
purified from yeast extracts using glutathione-agarose, and 400 ng of
the protein was assayed for its ability to induce p42
activation in Xenopus oocyte extracts as described in the
legend to Fig. 1.
Having determined that the Ste11 N-terminal
regulatory domain could partially block Ste20-induced p42 activation, we tested whether Ste11 was capable of inducing
activation of p42
in Xenopus oocyte extracts. A
full-length GST-Ste11 fusion protein (GSte11) was expressed in yeast
and purified from cell lysates (see ``Materials and
Methods''). As shown in Fig. 3B, GSte11 induced
p42
activation when added to Xenopus oocyte
extracts. While a Ste11 homolog has not yet been identified in X.
laevis, several MAPK and MAPKK homologs have been isolated from
this organism(32, 33, 34) . Our results
suggest the existence of a MAPK cascade in Xenopus oocytes
that strongly resembles the mating pheromone-responsive MAPK cascades
found in yeasts.
A mammalian Ste20/Shk1-related protein kinase,
Pak1, was previously purified as a Cdc42/Rac1-binding
protein(23) . We tested the ability of Pak1 to activate
p42 in Xenopus oocyte extracts. The addition of
a constitutively activated Pak1
N protein (residues 232-544)
to Xenopus oocyte extracts failed to induce activation of
p42
(Fig. 4). This result demonstrates that the
p42
cascade induced by Ste20 and Shk1 in Xenopus oocytes is selectively activated by a limited subset of PAKs. To
further examine the relationship between PAKs and the activation of
MAPK cascades, we determined whether PAKs induce activation of a
JNK/SAPK cascade in Xenopus oocytes. JNK/SAPKs comprise a
subfamily of stress-activated MAPKs distinct from the p42/p44 MAPK
subfamily(35) . As shown in Fig. 5, Pak1
N, as well
as the yeast Ste20 and Shk1 kinases, each induced activation of
JNK/SAPK in Xenopus oocyte extracts. MEKK, a potent activator
of JNK/SAPK in mammalian systems, also induced activation of JNK/SAPK
in Xenopus oocyte extracts (Fig. 5). In contrast,
JNK/SAPK activation was only weakly induced by H-Ras protein (Fig. 5).
Figure 4:
The mammalian Pak1 kinase does not induce
activation of p42 in Xenopus oocyte extracts.
GST-PAK1
N (GPak1
N) fusion protein was expressed and purified
from bacterial extracts as described under ``Materials and
Methods.'' Xenopus oocyte extracts were incubated with
either 1 µg of GPak1
N or 20 ng of GSte20
N, and
p42
activation was measured as described in the legend
to Fig. 1.
Figure 5:
Yeast and mammalian PAKs induce activation
of JNK/SAPK in Xenopus oocyte extracts. Xenopus oocyte extracts containing 2 µg of purified GST-JNK/SAPK were
incubated with 1 µg of GST, 1 µg of H-Ras[G12V], 20
ng of GSte20N, 3 µg of GMEKK, 40 ng of GShk1
N, 40 ng of
GShk1-FL, or 1 µg of GPak1
N-232 under conditions used
for p42
assays (see ``Materials and
Methods''). After 4 h, 10 µl of glutathione agarose beads was
added to each sample and incubated for 30 min at room temperature. The
beads were washed once with PBS and once with kinase buffer before
being resuspended in 20 µl of kinase buffer. GST-c-Jun
C
(residues 1-231) (2 µg) was added and the samples incubated
for 30 min at 30 °C. The reactions were stopped by adding 10 µl
of 3
SDS-PAGE sample buffer and boiling for 10 min. Samples
were resolved by electrophoresis in 4-20% gradient
SDS-polyacrylamide gels, then exposed to film for autoradiography.
Duplicate samples were blotted to nitrocellulose and probed with an
anti-GST antibody (Santa Cruz) and developed using ECL. JNK/SAPK
activation is expressed as the ratio of c-Jun
C phosphorylation
relative to c-Jun
C protein present as determined by scanning
densitometry. All values are expressed relative to GST, which was
normalized to a value of 1.0.
We have shown that yeast PAKs-Ste20 from S. cerevisiae and Shk1 from S. pombe trigger the activation of
p42 in cell-free extracts of X. laevis oocytes.
In contrast, we found that the mammalian kinase Pak1, a
Ste20/Shk1-related protein originally isolated as a Cdc42/Rac1-binding
protein(23) , did not induce p42
activation in Xenopus oocyte extracts. While not capable of inducing
activation of p42
, mammalian Pak1 did induce activation
of JNK/SAPK, as did the yeast Ste20 and Shk1 kinases. Our results
demonstrate that PAK-responsive MAPK cascades are conserved in
evolution and suggest that in higher organisms distinct MAPK modules
might be regulated by different types of PAKs. In particular, our
results suggest that protein kinases more closely related to Ste20 and
Shk1 than Pak1 might regulate p42
signaling pathways in
higher eukaryotes. We have, in fact, cloned rat cDNA sequences
corresponding to six distinct Ste20/Shk1-related protein kinases,
including Pak1 and Pak2. (
)One of these encodes a protein
kinase that is more closely related to Ste20 and Shk1 than Pak1 or
Pak2. It is possible that through evolution, distinct PAKs have evolved
to regulate distinct MAPK pathways. Given their capacity to activate
both p42
and JNK/SAPK pathways, it can be speculated
that the yeast Ste20 and Shk1 kinases represent ancestral forms of
PAKs.
While this manuscript was in preparation, other investigators provided evidence that in mammalian cells Cdc42 and Rac1 induce activation of JNK/SAPKs(36, 37, 38) . In one of these studies(38) , it was shown that the N-terminal regulatory domain of Pak1 blocked Cdc42 and Rac1-induced JNK/SAPK activation. However, since Pak1 binds directly to Cdc42 and Rac1(23) , it could not be concluded from this experiment whether Pak1 itself mediated Cdc42/Rac1-induced activation of JNK/SAPK. Our results demonstrate that Pak1 does indeed signal to the JNK/SAPK pathway. It is likely, therefore, that Pak1 represents a link between Cdc42/Rac1 and JNK/SAPK cascades.
It remains to be determined
exactly how PAKs fit into the regulatory networks that lead to the
activation of MAPK modules. In fission yeast, Shk1 and Cdc42 are
components of a Ras-dependent signaling complex that regulates both
cytoskeletal-dependent cellular morphology and a MAPK
module(20) . We have recently obtained genetic data suggesting
that Shk1 acts upstream of the MAPKKK, Byr2. Genetic data
also indicate that in S. cerevisiae Ste20 functions upstream
of Ste11(19) , which is both structurally and functionally
related to Byr2 (5) . Our results are consistent with
biochemical data from others showing that Ste20 immune complexes are
capable of phosphorylating Ste11 in vitro(21) . It is
possible that Ste20 and Shk1 are MAPKKK kinases, although direct
demonstration of this will require the use of purified proteins.
Recent studies have shown that in mammalian cells, as in fission
yeast, Cdc42 proteins participate in the regulation of
cytoskeletal-dependent cell morphology(39, 40) . The
structurally related small G proteins, Rac and Rho, also control
cytoskeletal organization(41, 42) . It is not yet
known whether Cdc42-activated protein kinases related to Ste20 and Shk1
participate in cytoskeletal regulation in mammalian cells. However, we
have obtained results consistent with this possibility. We have found
that expression of the N-terminal regulatory domain of Pak2 alters
cytoskeletal organization in Swiss 3T3 fibroblasts and disrupts normal
morphology in fission yeast. Thus, it appears likely that
in both yeasts and higher eukaryotes, Ste20/Shk1-related protein
kinases participate both in cytoskeletal regulation and in MAPK
signaling pathways. It remains to be determined whether there is a link
between MAPK signaling and control of the actin cytoskeleton. Further
study and characterization of PAKs using metazoan cell systems in
combination with yeast genetic and molecular biological approaches
should provide a better understanding of the roles these types of
protein kinases play in eukaryotic signal transduction pathways.