(Received for publication, May 4, 1995; and in revised form, July 27, 1995)
From the
We have isolated a novel member of the mammalian PAK (p21
activated kinase) and yeast Ste20 serine/threonine kinase family from
a mouse fibroblast cDNA library, designated mPAK-3. Expression of
mPAK-3 in Saccharomyces cerevisiae partially restores mating
function in ste20 null cells. Like other PAKs, mPAK-3 contains
a putative Cdc42Hs/Rac binding sequence and when transiently expressed
in COS cells, full-length mPAK-3 binds activated (GTPS (guanosine
5`-3-O-(thiotriphosphate)-bound) glutathione S-transferase (GST)-Cdc42Hs and GST-Rac1 but not GST-RhoA. As
expected for a putative target molecule, mPAK-3 does not bind to an
effector domain mutant of Cdc42Hs. Furthermore, activated His-tagged
Cdc42Hs and His-tagged Rac stimulate mPAK-3 autophosphorylation and
phosphorylation of myelin basic protein by mPAK-3 in vitro.
Interestingly, the amino-terminal region of mPAK-3 contains potential
SH3-binding sites and we find that mPAK-3, expressed in vitro and in vivo, shows highly specific binding to the SH3
domain of phospholipase C-
and at least one SH3 domain in the
adapter protein Nck. These results raise the possibility of an
additional level of regulation of the PAK family in vivo.
The cellular functions of various members of the Rho subclass of GTP-binding proteins, including RhoA, Rac1, and Cdc42Hs, have recently received a great deal of attention and are thought to be essential for a number of changes in the actin cytoskeleton (Kozma et al., 1995; Nobes and Hall, 1995) (for reviews, see Hall(1994) and Chant and Stowers(1995)). Recent reports suggest that these three GTP-binding proteins participate in a hierarchical series of cytoskeletal-mediated events, starting with the generation of filopodia by activated Cdc42Hs and followed by the successive appearance of lamellipodia, stimulated by activated Rac1, and actin stress fibers, stimulated by activated RhoA (Kozma et al., 1995; Nobes and Hall, 1995). Despite the indication that these different events are coordinated and potentially the outcome of a single signaling pathway (Cdc42Hs-Rac1-RhoA), it also has been shown that different extracellular signals promote the individual activation of Cdc42Hs, Rac1, and RhoA. In the case of Swiss 3T3 fibroblasts, these signals are bradykinin, platelet-derived growth factor, and lysophosphatidic acid, respectively. The challenging problems that remain are to understand the detailed biochemical events that give rise to these cytoskeletal changes and to determine how different inputs can elicit specific effects through the Cdc42Hs, Rac1, and RhoA GTP-binding proteins.
A logical starting place for
obtaining biochemical and mechanistic information would be studies of
the regulation of the GTP-binding/GTPase cycles of these Rho subclass
proteins. In fact, a significant amount of information is available
regarding the actions of three classes of regulators, the GDP
dissociation inhibitors, the GEFs ()(guanine
nucleotide-exchange factors), and the GAPs (GTPase-activating
proteins). The GEFs and GAPs have been especially interesting because
they represent families of molecules that have in one way or another
been implicated in cell growth regulation. For example, the prototype
Rho subclass GEF is the oncoprotein Dbl, which contains a region of
250 amino acids that is essential both for its transforming and
guanine nucleotide exchange activity (Hart et al., 1994). This
region, designated the Dbl homology domain, is found in a number of
other proteins including the Vav, Ect2, and Ost oncoproteins (Katzav et al., 1989; Miki et al., 1993; Horii et
al., 1994), as well as in the Tiam-1 protein that has been
implicated in metastasis (Habets et al., 1994) and the Fgd-1
protein that is involved in the faciogenital dysplasia developmental
disorder (Pasteris et al., 1994). Likewise, the Cdc42Hs GAP
shares a region of homology with a number of potential growth
regulatory proteins including Bcr (Diekmann et al., 1991), the
Ras GAP-associated protein p190 (Settleman et al., 1992), the
Abl SH3-binding protein 3BP-1 (Cicchetti et al., 1992), and
the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (Otsu et al., 1991). The interactions of Dbl and these different
GAPs with Cdc42Hs and related GTP-binding proteins have been studied in
detail and point to a very tight regulation of the GTP binding/GTPase
cycles of the Rho subclass proteins in order to ensure normal cell
growth and developmental processes.
Despite the increased
understanding of the mechanisms underlying the regulation of their
GTPase cycles, it is not clear how Cdc42Hs, Rac1, or RhoA mediate the
observed cytoskeletal changes. Clearly, an important step will be to
identify and characterize the effector proteins that are directly
responsible for mediating the actions of the GTP-bound forms of
Cdc42Hs, Rac1, and RhoA. Thus far, two putative targets have been
identified for the Cdc42Hs and Rac1 proteins. One is the 85-kDa
regulatory subunit of the PI 3-kinase (Zheng et al., 1994).
The GTP-bound forms of the Cdc42Hs and Rac1 proteins have been shown to
bind to the GAP homology domain of p85 and to elicit a 4-5-fold
stimulation of PI 3-kinase activity. The GTP-bound form of Ras also
binds to the PI 3-kinase, through a direct interaction with its 110-kDa
catalytic domain (Rodriguez-Viciana et al., 1994). Thus, the
PI 3-kinase may serve as a point of convergence for Cdc42Hs or Rac1 and
Ras, enabling these different GTP-binding proteins to cooperate in the
stimulation of cytoskeletal changes that accompany growth factor
binding to receptors. A second, recently identified potential target
for the Cdc42Hs and Rac1 proteins is a 65-kDa serine/threonine protein
kinase called PAK (p21 activated kinase) that was reported to be the
mammalian homolog of the Saccharomyces cerevisiae Ste20 kinase
(Manser et al., 1994). The involvement of Ste20 in the
pheromone/mating factor pathway in yeast has been well documented, and
in fact the complete signaling pathway starting with the mating factor
receptor and continuing through a protein kinase cascade to the nucleus
has been elucidated (see Herskowitz(1995) for review). However, at the
present time, much less is known about the actions of the mammalian PAK
or the effects of Cdc42Hs (or Rac1) stimulation of this kinase and how
this stimulation impacts on the cytoskeleton. It now seems likely that
a family of mammalian PAKs exist, and in the present report we describe
a new member of this kinase family that was initially identified using
a Cdc42Hs-GTPS fusion protein as an affinity reagent for detection
of cellular targets. This serine/threonine kinase is
80% identical
to other PAK/Ste20 kinases and is designated mPAK-3. We show that
mPAK-3 can complement deletions of the Ste20 gene product in S.
cerevisiae and that its kinase activity toward exogenous
phosphosubstrates is strongly stimulated by GTP-bound Cdc42Hs and Rac1.
Moreover, mPAK-3 shows a high degree of binding specificity for a
particular group of SH3 domains, which may be involved in the
regulation and localization of this new protein kinase.
Lipofectamine-mediated transient transfections of
COS cells were performed according to the manufacturer's protocol
(Life Technologies, Inc.). Briefly 2-3 10
COS
cells were plated in 35-mm dishes 18-24 h prior to transfection.
0.5 µg of J3HmPAK-3 DNA and 5 µl of Lipofectamine reagent were
added to each plate in 1 ml of Dulbecco's modified Eagle's
medium (DMEM) in the absence of serum. After 5 h, 1 ml of DMEM
containing 20% fetal calf serum (Life Technologies, Inc.) was added;
after 14-18 h, the medium was replaced with fresh DMEM containing
10% fetal calf serum. Cells were lysed 48-72 h after addition of
DNA.
Figure 1:
A protein kinase
activity associates with GST-Cdc42-GTPS. A, NIH 3T3 cell
lysates were incubated with immobilized GST (lanes1, 4, 7, and 10), GST-Cdc42Hs-GDP (lanes2, 5, 8, and 11), and
GST-Cdc42Hs-GTP
S (lanes3, 6, 9, and 12) for 1 h at 4 °C. Bound proteins were washed and subjected to an in vitro kinase assay
in the absence (lanes 1-3) or presence of exogenous
substrates, i.e. myelin basic protein (lanes
4-6),
-casein (C, lanes7-9) and histone H1 (H, lanes
10-12). The band at 45 kDa corresponds to GST-Cdc42, which
is phosphorylated in the absence of an exogenous substrate. B,
NIH 3T3 cell lysates were incubated with immobilized GST,
GST-Cdc42Hs-GDP, and GST-Cdc42Hs-GTP
S (lanes3, 4, and 5, respectively) for 1 h at 4 °C. Lane1 contains 5.7% of the whole cell lysate (WCL)
used in the binding reactions. Bound proteins were washed, separated on
SDS-PAGE, and transferred to an Immobilon P membrane. The bottom part
of the gel (<50 kDa) was stained with Coomassie Blue to ascertain
that each lane contained equal amounts of fusion protein. The proteins
on the membrane were subjected to a kinase assay using
[
-
P]ATP and MnCl
as described
under ``Experimental Procedures.'' Autoradiography was for 14
h at room temperature. E. coli-expressed, immobilized
GST-Cdc42Hs-GDP was incubated with lysis buffer (LB) as a
negative control in lane2. Phosphoamino acid
analysis was performed on the band in lane5 as
described under ``Experimental Procedures.'' PS, PT, and PY indicate the position of nonradioactive
phosphoserine, phosphothreonine, and phosphotyrosine
markers.
Figure 2: Amino acid sequence comparison of rat p65PAK and mPAK-3. The amino acid sequence of mPAK-3 was deduced from the sequence of a cDNA clone isolated from a mouse fibroblast cDNA library. Protein sequences are presented in single-letter code. The sequences were aligned using the GAP program. Underlined residues in mPAK-3 indicate putative SH3-binding regions. mPAK-3 shows 81% identity and 89% similarity to rat p65PAK. Romannumerals indicate conserved kinase subdomains. Dashes between amino acids represent identical sequences, doubledots signify conservative changes, and singledots denote less conservative changes. A stretch of acidic residues is highlighted in bold. The putative Cdc42 and Rac binding domain of mPAK-3 (amino acids 65-128) shares similarity with rat p65 PAK.
There are at
least three mammalian PAK family members. Two human PAK family members
have recently been identified. One, designated hPAK-1, is 98% identical
to the rat p65PAK and is the human homolog of rat p65PAK, while the
second, hPAK-2, is 78% identical to rat p65PAK. The mouse
homolog that we have identified is 81% identical to hPAK-1 and 76%
identical to hPAK-2, and so we believe it represents a third mammalian
form and have designated it mPAK-3. The kinase domains and the putative
Cdc42Hs/Rac binding domains are highly conserved between the three
PAKs. However, the amino terminus of mPAK-3 diverges from that of
p65PAK and the two human proteins. In addition, mPAK-3 contains four
distinct proline-rich sequences that represent potential binding sites
for SH3 domain-containing proteins (Feng et al., 1994), which
are also present in hPAK-1, and a stretch of acidic amino acids
(residues 173-185), which is not fully conserved in other members
of the family.
Figure 3: mPAK-3 can complement a Ste20 defect in yeast. S. cerevisiae strains (described under ``Experimental Procedures'') bearing a deletion in ste20, ste5, or ste11 were transformed with vector alone (pYES2), mPAK-3, ste5, ste11, and ste20 plasmids, as indicated, were grown on minimal medium lacking uracil with galactose as the carbon source followed by patch mating to strain RSY16. Diploids were selected for growth on minimal medium lacking leucine.
Figure 4:
mPAK-3 specifically binds Cdc42Hs and
Rac1. COS cells were transiently transfected with a plasmid encoding
NH-terminal HA-tagged mPAK-3 using lipofectamine. After 48
h, cells were lysed and lysates incubated with immobilized GST, wild
type GST-Cdc42Hs-GDP, GST-Cdc42Hs-GTP
S, GST-Rac1-GDP,
GST-Rac1-GTP
S, GST-RhoA-GDP, and GST-RhoA-GTP
S, a putative
effector domain mutant GST-Cdc42HsT35A-GDP, GST-Cdc42HsT35A-GTP
S,
and a GTPase-defective mutant GST-Cdc42HsQ61L-GTP
S in lanes3-11, respectively, for 1 h at 4 °C. Lane1 represents 19% of the whole cell lysate (WCL)
used in the binding reaction. Bound proteins were Western-blotted and
probed with anti-HA (mAb 12CA5) to detect HA-mPAK-3. The higher
molecular weight band seen in lane1 (here and in Fig. 6B) is a nonspecific band that cross-reacts with
the anti-HA antibody and is also observed in untransfected COS
cells.
Figure 6:
mPAK-3 binds to the PLC- and Nck SH3
domains. A, in vitro translated
[
S]methionine-labeled mPAK-3 was incubated with
approximately equal amounts of immobilized GST(-) or GST fusion
proteins (GST-SH3 affinity precipitates (AP)) as indicated. Lane1 represents 20% of the reticulocyte lysate (input). After washing resin-bound proteins were analyzed by
SDS-PAGE and fluorographed. B, HA-tagged mPAK-3 from
transiently transfected COS cell lysates were incubated with
immobilized GST(-) or GST fusion proteins as indicated. Lane1 represents 10% of the whole cell lysate (wcl)
used in the binding reactions. After washing, resin-bound proteins were
Western blotted and probed with anti-HA
antibody.
Treatment of
affinity-precipitated Cdc42Hs-GTPS/mPAK-3 complexes with potato
acid phosphatase eliminated the retarded electrophoretic mobility band
observed in Fig. 4, and a band appeared that had faster mobility
than mPAK-3 in lysates (data not shown). These results suggest that
mPAK-3 has a certain basal level of phosphorylation in cell lysates and
that binding to GTP-bound forms of Cdc42Hs further increases the level
of phosphorylation, for example by stimulating the autophosphorylation
activity of the kinase and causing the electrophoretic mobility shift.
Figure 5:
In vitro activation of mPAK-3. A, COS cells were transfected with HA-mPAK-3 and lysed as
described under ``Experimental Procedures.'' Anti-HA
immunoprecipitates were subjected to an in vitro kinase assay
using [-
P]ATP and 5 mM MgCl
in the presence of GDP- or GTP
S-bound Cdc42Hs (lanes1, 2, 8, and 9) or GDP- or
GTP
S-bound Rac1 (lanes3, 4, 6, and 7), fused to either GST or a hexahistidine
(His) tag. Reactions were stopped after 5 min with 2
SDS sample
buffer. B, procedures were as described in A except that units corresponding to equivalent amounts of
GTP
S-bound His-Cdc42Hs or His-Rac1 were added in each lane as
indicated. A unit is determined by [
S]GTP
S
counts bound to the G-protein. His-Cdc42Hs-GDP was used in lane 12 (control).(-) indicates immunoprecipitates
alone.
In this report we describe a third member of the mammalian
family of PAK serine/threonine kinases. This protein, mPAK-3, is
80% identical to both rat PAK (i.e. the first member of
the family identified by Manser and colleagues (Manser et al.,
1994)) and the human PAK-1,
and is
76% identical to a
second human homolog, hPAK-2.
The mPAK-3 also is
70%
identical to the catalytic domain of S. cerevisiae protein,
Ste20, and we show here that mPAK-3 will compensate for mating defects
caused by the deletion of ste20. It has been well established
that the Ste20 kinase is a key participant in the pheromone mating
factor pathway. Specifically, in response to pheromone, Ste20 initiates
a protein kinase cascade that includes Ste11 (the functional homolog of
mammalian mitogen-activated protein (MAP) kinase kinase kinases or
MEKKs (Lange-Carter et al., 1993)), Ste7 (the homolog of
mammalian MAP kinase kinases or MEKs (Crews et al., 1992;
Ashworth et al., 1992)), and FUS3/KSS1 (the homolog of
mammalian MAP/ERK kinases). (For recent reviews on the MAP kinase
cascade, see Herskowitz(1995), Marshall(1994), Johnson and
Vaillancourt(1994), and Errede and Levin(1993).) Given this role of
Ste20 in yeast, it will be important to see if the different mammalian
PAKs initiate kinase cascades involving MEKKs or MEK proteins in
response to extracellular signals. In particular, since mPAK-3 is
activated by activated Rac1 and Cdc42Hs, it may initiate kinase
cascades leading to mitogenic responses to growth factors.
Extracellular signals, including bradykinin, PDGF, and lysophosphatidic
acid, have indirectly been shown to activate Cdc42Hs and Rac1 (Kozma et al., 1995; Ridley et al., 1992; Ridley and Hall,
1992). In addition, Rac1 has recently been shown to act downstream of
Ras during Ras-induced cellular transformation and to possess
growth-stimulatory properties (Qiu et al., 1995).
Alternatively, the activation of MAP kinases by mPAK-3 could mediate
the characteristic cytoskeletal re-arrangements induced by Cdc42Hs,
Rac1, and their respective extracellular stimuli.
The
serine/threonine kinase activity of mPAK-3 is strongly stimulated by
both the Cdc42Hs and Rac1 proteins, but not by RhoA. This is similar to
what was observed for the rat p65PAK (Manser et al., 1994).
The mechanism that underlies the stimulation of kinase activity by
these GTP-binding proteins is not yet known, although it is likely that
the binding of the GTP-binding protein to a specific region within the
amino-terminal half of the kinase releases a negative constraint.
De-repression of PAK activity by Cdc42Hs and Rac1 would therefore
represent another example of the common mechanism of activation of
several protein kinases. Such an activation mechanism would predict
that the binding of a GTP-binding protein to a PAK would simultaneously
result in the stimulation of the kinase activity. However,
interestingly, we have found that while GST-Rac1, when in the
GTPS-bound state, will bind specifically to mPAK-3, it shows no
detectable stimulation of the mPAK-3 kinase activity. Thus, this
represents an example where specific binding to mPAK-3 by a GTP-binding
protein is uncoupled from the stimulation of kinase activity and
suggests that the activation mechanism entails more than simply a
single (specific) binding event. Another Rac1 fusion protein, that
contains 20 amino acids upstream from the start site for Rac1 (i.e. a His-tagged Rac1), exhibits both specific binding and stimulation
of kinase activity. At present, we are trying to understand the
molecular mechanism that underlies the striking differences observed
with the different Rac fusion proteins, since the results indicate that
the presence of the GST moiety interferes with a second type of
interaction between the GTP-binding protein (Rac) and the kinase that
is necessary for the stimulation of kinase activity. Apparently, the
presence of the GST moiety does not interfere with this second
stimulatory interaction between Cdc42Hs and mPAK-3, since GST-Cdc42Hs
both specifically binds to and stimulates mPAK-3.
When comparing the dose-dependent stimulation of mPAK-3 by the His-tagged Cdc42Hs and Rac1 proteins, we find that these two GTP-binding proteins are approximately equipotent in activating mPAK-3. This then raises the question of GTP-binding protein/target specificity. Do both of these GTP-binding proteins bind to the same target in the cell or do the two GTP-binding proteins in fact regulate different members of the mammalian PAK family? Both Cdc42Hs and Rac1 have been implicated in cytoskeletal organization (Kozma et al., 1995; Nobes and Hall, 1995; Ridley et al., 1992), and recent studies even argue that Cdc42Hs may be functioning upstream from Rac1 in a common pathway that impacts on the cytoskeleton (Kozma et al., 1995; Nobes and Hall, 1995). Thus, one possibility is that Cdc42Hs may initially activate a specific PAK to initiate cytoskeletal alterations. However, this stimulation may be transient and perhaps is replaced by a more persistent stimulation of the same PAK when Rac1 is subsequently activated. Another possibility is that the Cdc42Hs- and Rac1-mediated stimulations of PAKs (either the same PAK family member or distinct members) occur with both spatial and temporal specificity and that this specificity accounts for distinct cytoskeletal events (e.g. filopodia formation in the case of activated Cdc42Hs versus membrane ruffling in the case of activated Rac1). Presumably, such specificity would be mediated by other cellular proteins.
One possibility for additional modes of
regulation of PAK activity is via the binding of SH3 domain-containing
proteins. mPAK-3 contains four potential (PXXP) SH3
domain-binding motifs within the amino-terminal half of the molecule.
Moreover, we have found through in vitro binding assays that
mPAK-3 binds with high specificity to the SH3 domains of PLC and
Nck. To our knowledge this represents the first example of an
identified serine/threonine protein kinase that is able to associate
with SH3 domains. An unidentified serine kinase activity has recently
been shown to bind to Nck via one of its SH3 domains (Chou and
Hanafusa, 1995); based on our results, this could be a PAK family
member. The Btk tyrosine kinase has been shown to bind in vitro to the SH3 domains of Fyn, Lyn, and Hck (Cheng et al.,
1994). One of the SH3-binding sites within Btk is strikingly similar to
one of the potential SH3-binding sites in mPAK-3 (residues 33-41,
KPLPXXPEE), raising the possibility that these two otherwise
quite distinct protein kinases share common regulatory mechanisms.
Interestingly, another target of Cdc42Hs, the 85-kDa regulatory subunit
(p85) of the PI 3-kinase, also possesses SH3-binding sites. PLC-
has been shown to associate with the actin cytoskeleton in fibroblasts
(McBride et al., 1991) and may therefore serve to localize
mPAK-3 to sites of Cdc42Hs/Rac1 action. Furthermore, the substrate of
PLC-
, phosphatidylinositol 4,5-bisphosphate, may play a role in
actin polymerization-depolymerization events via an interaction with
the actin-binding protein profilin (Goldschmidt-Clermont et
al., 1990). Thus, mPAK-3 may serve to connect cytoskeletal
interactions involving PLC-
and/or phosphatidylinositol
4,5-bisphosphate with Cdc42Hs or Rac1 activation. The adapter protein
Nck, which contains one SH2 and three SH3 domains, is recruited to
activated PDGF receptors via its SH2 domain (Nishimura et al.,
1993). Nck could therefore mediate localization of mPAK-3 to its sites
of action in response to growth factors such as PDGF, which may also,
through other pathways, activate Cdc42 and/or Rac1. It will be
important to determine whether mPAK-3 associates with PLC-
or Nck in vivo, and, if so, whether this association is modulated in
response to extracellular stimuli. Additional studies will be directed
toward determining the specific proline-rich region on mPAK-3 that is
responsible for these interactions in order to generate and express
mutant mPAK-3 proteins that may be used to uncouple specific cellular
events that normally require the convergence of GTP-binding proteins,
mPAK-3, and SH3 domain-containing proteins.