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Rho Family GTPases Regulate p38 Mitogen-activated Protein Kinase through the Downstream Mediator Pak1


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The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity. p21-activated kinase 1 (Pak1) is a potential mediator of Rac/Cdc42 signaling, and we observe that Pak1 stimulates p38 activity. A dominant negative Pak1 suppresses both interleukin-1- and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK.
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Rho Family GTPases Regulate
p38 Mitogen-activated Protein
Kinase through the Downstream
Mediator Pak1*
(Received for publication, July 25, 1995, and in revised form,
August 17, 1995)
Shengjia Zhang‡§, Jiahuai Han‡, Mary Ann
Sellsi, Jonathan Chernoffi, Ulla G. Knaus‡§,
Richard J. Ulevitch‡, and Gary M. Bokoch‡§
From the Departments of Immunology and §Cell
Biology, Scripps Research Institute, La Jolla, California
92037 and the iFox Chase Cancer Center,
Philadelphia, Pennsylvania 19111
The stress-activated p38 mitogen-activated protein
(MAP) kinase defines a subgroup of the mammalian
MAP kinases that appear to play a key role in regulating
inflammatory responses. Co-expression of constitutively
active forms of Rac and Cdc42 leads to activation of p38
while dominant negative Rac and Cdc42 inhibit the abil-
ity of interleukin-1 to increase p38 activity. p21-acti-
vated kinase 1 (Pak1) is a potential mediator of Rac/
Cdc42 signaling, and we observe that Pak1 stimulates
p38 activity. A dominant negative Pak1 suppresses both
interleukin-1- and Rac/Cdc42-induced p38 activity. Rac
and Cdc42 appear to regulate a protein kinase cascade
initiated at the level of Pak and leading to activation of
p38 and JNK.
Rac and Cdc42 are members of the Rho family of small
guanosine 59-triphosphate (GTP)-binding proteins. These
GTPases regulate assembly of actin cytoskeletal structures
associated with cell motility and metastasis, as well as the
generation of bactericidal oxygen metabolites by the phagocyte
NADPH oxidase (1, 2). Rac was also shown to be an important
component of cellular transformation by Ras oncogenes, al-
though the mechanisms by which Rac contributes to the trans-
formation process are unknown (3). Regulation of nuclear sig-
naling by Rho family GTPases has recently been described (4),
possibly through their stimulatory effects on c-Jun amino-ter-
minal kinase (JNK)
(5, 6).
JNKs or stress-activated protein kinases represent a second
class of the mammalian mitogen-activated protein (MAP) ki-
nases, which includes the “classical” extracellular signal-regu-
lated kinases (ERK) (7, 8). An additional class, which presents
substantial similarity to the Saccharomyces cerevisiae HOG1
kinase involved in responses to increased extracellular osmo-
larity (reviewed by Herskowitz (9)), is p38 MAP kinase. Like
HOG1, p38 can be activated by changes in osmolarity but also
appears to participate in the inflammatory response to lipo-
polysaccharides or to inflammatory mediators such as interleu-
kin-1 (IL-1) or tumor necrosis factor (10–12). The mechanisms
by which p38 activation occurs in response to external stimuli
remain to be determined. Induction of p38 activity by IL-1 or
tumor necrosis factor
has little effect on ERK activity, sug-
gesting upstream signaling via Ras does not play an important
role in p38 activation.
In their active GTP-bound forms, both Rac and Cdc42 bind to
and stimulate the activity of a group of 65–68-kDa Ser/Thr
kinases in mammalian cells (13–15). These p21-activated ki-
nases (Paks) are homologous to the yeast Ste20 kinase involved
in regulating yeast MAP kinase cascades controlling the mat-
ing pheromone response pathway, invasive growth of haploid
yeast, and pseudohyphal differentiation in diploid yeast (9). As
in the yeast mating factor pathway, we have recently estab-
lished that Pak activity can be regulated by mammalian G
protein-coupled receptors through a pertussis toxin-sensitive G
protein (15). In the present communication, we show that Pak
and its upstream regulators, Rac and Cdc42, couple to and
regulate the activity of p38 MAP kinase and are an integral
part of the signaling pathway linking cell surface proinflam-
matory receptors to p38 activation.
Expression Plasmids—The Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-
Asp-Lys; Immunex, Seattle, WA) was inserted between codons 1 and 2
of the p38 and JNK cDNAs by insertional overlapping polymerase chain
reaction (16) and placed into the pcDNA3 expression vector (Invitrogen)
to generate the plasmids pcDNA3-Flag-JNK1 and pcDNA3-Flag-p38
MAP kinase. Hemagglutinin (HA)-tagged Rac1(T17N), HA-
Rac1(Q61L), HA-Cdc42Hs(Q61L), and HA-Cdc42Hs(T17N), were all
inserted into the polylinker region of the pcDNA3 vector. HA-
RhoA(Q63L) and HA-RhoA(T19N) were inserted into the pCMV5 vector
(17), HA-Pak1 into the pJ3H expression vector (18), and c-Myc-tagged
ERK1 into the pJ3M vector (18). HRas(Q61L) and Raf 22W (an amino-
terminal-truncated active form) (19) were in the pZip-neo-svx(1) vector
and were provided by J. Jackson (Scripps Research Institute).
Transient Cell Expression—COS-7 and HeLa cells were maintained
in Dulbecco’s modified Eagle’s medium supplemented with 5% bovine
serum. Cells on 35-mm plates were transiently transfected with 1
each plasmid DNA (see below) using Lipofectamine reagent (Life Tech-
nologies, Inc.) according to the manufacturer’s recommendations.
Transfection efficiency was evaluated using a luciferase co-transfection
assay (Promega). After 48 h, the cells were treated with or without UV
radiation or IL-1 as described (12, 20). Cells were solubilized with lysis
buffer (25 mMHepes, pH 7.6, 3 mM
-glycerophosphate, 3 mMEDTA, 3
mMEGTA, 250 mMNaCl, 1% Nonidet P-40, 1 mMdithiothreitol, 0.1 mM
orthovanadate, 1 mMphenylmethylsulfonyl fluoride, 10
g/ml leupep-
tin, 0.078 trypsin inhibitory units/ml aprotinin) for 30 min with shaking
at 4 °C and then cleared by centrifugation at 100,000 3gfor 30 min at
4 °C prior to immunoprecipitation and kinase assay. Expression levels
of cDNA constructs after transient transfection were verified using the
respective epitope tag antibodies.
Kinase Assays—Mouse monoclonal antibodies against the Flag
epitope, M2 (Kodak Scientific Imaging Systems), the c-myc epitope,
9E10 (Santa Cruz Biotechnology), HA epitope, and 12CA5 (kindly pro-
vided by I. Wilson, Scripps Research Institute) or rabbit polyclonal
Pak1 antibody (15) were prebound to protein G-Sepharose or protein
A-Sepharose beads, respectively. 20
l of a 1:1 suspension of beads was
added to 300-
l cell lysates and gently shaken for3hat4°C.The
precipitates were washed 6 times with 1 ml of wash buffer containing 25
* This work was supported by National Institutes of Health Grants
HL48008 and GM39434 (to G. M. B.), AI35947 (to U. G. K.), AI15136
and GM37696 (to R. J. U.), GM51471 (to J. H.), and CA58836 (to J. C.),
by University of California Breast Cancer Research Program Grant
1IB-0491 (to U. G. K.), and by a grant from the W. W. Smith Foundation
(to J. C.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
To whom correspondence should be addressed: Depts. of Immunol-
ogy and Cell Biology, IMM14, Scripps Research Inst., 10666 N. Torrey
Pines Rd., La Jolla, CA 92037.
The abbreviations used are: JNK, c-Jun amino-terminal kinase;
ERK, extracellular signal-regulated kinase; MAP, mitogen-activated
protein; IL, interleukin; Pak, p21-activated kinase; HA, hemagglutinin.
Vol. 270, No. 41, Issue of October 13, pp. 23934–23936, 1995
© 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
by guest on September 15, 2015 from
mMHepes, pH 7.6, 50 mMNaCl, 0.1 mMEDTA, 0.05% Triton X-100, 0.1
mMsodium orthovanadate, 1 mMphenylmethylsulfonyl fluoride, 10
g/ml leupeptin, and 0.078 trypsin inhibitory units/ml aprotinin prior
to kinase assay. Immunocomplex kinase assays were performed at
30 °C for 20 min using 30
l of kinase buffer (20 mMHepes, pH 7.6, 20
-glycerophosphate, 0.1 mMsodium orthovanadate, 2
mMdithiothreitol), 3–5
g of substrate, 20
MATP, and 10
Ci of
P]ATP per assay. Substrates utilized were: a glutathione S-trans-
ferase amino-terminal truncated (amino acids 1–109) activating tran-
scription factor-2 (20) for p38, glutathione S-transferase-c-Jun for JNK
(21–23), and myelin basic protein for ERK1 and Pak1. The incubation
was terminated with 20
lof43Laemmli sample buffer, and then the
products were resolved by SDS-polyacrylamide gel electrophoresis on
12% gels and visualized by autoradiography.
The proinflammatory cytokine IL-1 is a physiological regu-
lator of p38 (12), causing a marked and rapid stimulation of p38
activity in HeLa cells (Fig. 1). We observed that IL-1 also
stimulated Pak1, with Pak1 activation slightly preceding that
of p38 (Fig. 1). Since Rac and Cdc42 are known regulators of
Pak1 (13–15), we speculated that IL-1 might be linked to Pak1
activation through these GTPases and that this pathway might
be involved in regulation of p38. In support of this hypothesis,
we observed that expression of dominant negative forms of both
Rac and Cdc42 effectively inhibited the ability of IL-1 to stim-
ulate p38 activity (Fig. 2). Inhibition was directly dependent
upon the amount of the dominant negative plasmid used.
While dominant negative forms of Rac and Cdc42 inhibited
p38 activation by IL-1, we wanted to determine whether Rac
and Cdc42 were sufficient to stimulate p38 activity. Co-expres-
sion of active Rac or Cdc42 with p38 in COS cells caused a large
enhancement of p38 activity, comparable with that seen with
stimulation by UV radiation, which maximally activates the
enzyme and which serves an indicator of the total levels of p38
expressed and present in the immune precipitates (Fig. 3, A
and B). Expression of wild type Rac had only a slight effect on
p38 activity (data not shown). This effect was specific for the
GTPases Rac and Cdc42, as we failed to observe stimulation
when activated forms of H-Ras, Raf, or RhoA were co-trans-
fected with p38 (Fig. 3C).
The role of Pak in the p38 activation process was also as-
sessed. Co-expression of wild type Pak1 itself with p38 caused
a marked increase in p38 activity (Fig. 3, Aand B). Pak1
appears to become activated when expressed in a COS cell
environment, possibly due to the presence of low levels of active
GTP-bound Cdc42.
However, when we co-expressed Pak1 with
constitutively GTP-bound Rac or Cdc42, we observed a greater
increase in p38 activity, indicating that the action of Pak could
be enhanced by these known activators of the enzyme’s cata-
lytic function.
We utilized a Pak1 containing a single point mutation
(K299R) in the kinase domain, which renders the enzyme cat-
alytically inactive (24), to investigate the role of Pak1 in p38
S. Zhang and G. M. Bokoch, unpublished observations.
FIG.1.Time course of p38 MAP kinase and Pak1 activation by
IL-1. Epitope-tagged p38 MAP kinase or Pak1 was expressed in HeLa
cells as described under “Experimental Procedures.” After 48 h, the
cells were treated with 10 ng/ml IL-1
(Genzyme Corp.) for the indi-
cated time periods at 37 °C prior to immunoprecipitation with Flag or
Pak1 antibody, respectively, and kinase assay. Phosphorylated myelin
basic protein and activating transcription factor-2 were detected after
12% SDS-polyacrylamide gel electrophoresis by autoradiography and
quantitated using PhosphorImager and ImageQuant software (Molec-
ular Dynamics). Open bars represent p38 activity and solid bars Pak1
activity. The data presented represent the relative kinase activity
quantified from a single experiment representative of two; the autora-
diograph from this experiment is shown in the inset.
FIG.2.Inhibition of IL-1-stimulated p38 MAP kinase activity
by dominant negative forms of Rac1, Cdc42, and Pak1. Epitope-
tagged p38 MAP kinase in pcDNA3 vector (0.2
g/plate) was transiently
transfected into HeLa cells together with either the dominant negative
form of Rac1 (top panel), Cdc42 (middle panel), or Pak1 (bottom panel).
The molar ratio of the dominant negative plasmids to p38 cDNA,
respectively, is indicated at the bottom of the figure, with the total DNA
concentration kept constant by supplementation with pcDNA3 vector.
No dom.neg. indicates the IL-1-activated control in the absence of any
dominant negative DNA. Expression of p38 was similar under each
condition as determined by Western blotting (not shown). The cells
were stimulated 48 h after transfection with 10 ng/ml IL-1 for 30 min at
37 °C, and then p38 MAP kinase activity was measured. Results are
representative of two similar experiments.
FIG.3.Stimulation of p38 MAP kinase by Rho family GTPases
acting through Pak. Epitope-tagged p38 MAP kinase was co-ex-
pressed in COS-7 cells with the following cDNAs and then immunopu-
rified after 48 h and assayed for kinase activity. The total DNA concen-
tration in each condition was maintained constant by supplementation
with pcDNA3 vector. Results are representative of two or more exper-
iments. A, pcDNA3-p38 and: lanes 1 and 5,1empty pcDNA3 vector;
lanes 2 and 6,1pJ3H-Pak1; lanes 3 and 7,1pcDNA3-Rac1(Q61L);
lanes 4 and 8,1pcDNA3-Rac1(Q61L) and pJ3H-Pak1. B, pcDNA3-p38
and: lanes 1 and 5,1empty pcDNA3 vector; lanes 2 and 6,1pJ3H-
Pak1; lanes 3 and 7,1pcDNA3-Cdc42(Q61L); lanes 4 and 8,1
pcDNA3-Cdc42(Q61L) and pJ3H-Pak1. C, pcDNA3-p38 and: lanes 1
and 5,1empty pcDNA3 vector; lanes 2 and 6,1pZip-neo-HRas(Q61L);
lanes 3 and 7,1pZip-neo-Raf*(22W); lanes 4 and 8,1pCMV5-
RhoA(Q63L). D, pcDNA3-p38 and: lanes 1 and 6,1pJ3H-Pak1(K299R)
alone; lanes 2 and 7,1pcDNA3-Rac1(Q61L); lanes 3 and 8,1
pcDNA3-Rac1(Q61L) and Pak1(K299R) at a 1:10 DNA ratio); lanes
4and 9,1pcDNA3-Cdc42(Q61L); lanes 5 and 10,1
pcDNA3-Cdc42(Q61L) and Pak1(K299R) at a 1:10 DNA ratio. Each
condition is also shown after stimulation with UV light (lanes 5–8 of
panels A–C and lanes 6–10 of panel D) to assess maximal stimulation of
p38 activity and p38 expression under each condition.
p21-activated Kinases Regulate p38 Activity 23935
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activation by Rac and Cdc42. This construct behaves as a
dominant negative inhibitor of Pak activity in vivo,
but does
not appear to act merely by titrating out Rac and/or Cdc42, as
it does not inhibit Rac-potentiated cell transformation.
inant negative Pak inhibited nearly all of the p38 stimulatory
capability of Rac and Cdc42, suggesting that the effects of both
of these GTPases on p38 were mediated via Pak activation (Fig.
3D). In studies not shown we observed that UV-induced p38
activation can be blocked by increasing the amount of plasmid
DNAs encoding Pak1 (K299R) used in the co-transfection assay
using ratios similar to those shown in Fig. 2. Additionally, Pak1
(K299R) was able to effectively block activation of p38 by IL-1.
Taking into account the ability of IL-1 to stimulate Pak1 activ-
ity with a similar time course as that for p38 activation, the
ability of a dominant negative Pak1 to block p38 activation by
IL-1, and the ability of Pak1 itself to stimulate p38 activity, we
conclude that the activity of Pak1, regulated by the upstream
GTPases Rac and/or Cdc42, is an integral component of the
signaling process linking cytokine receptors to p38 activation.
The regulatory effects of Pak1 are not limited to the p38
pathway. The JNKs form an additional branch of the mamma-
lian MAP kinase family, which are regulated by many of the
same upstream stimuli as p38 (7–9). In addition to the recently
reported ability of Rac and Cdc42 to stimulate JNK activity (5,
6), we observed that Pak1 could activate JNK activity as well
(data not shown). In contrast, we could detect no stimulatory
effect of activated Rac, Cdc42, or Pak1 on the ERK branch of
the MAP kinase family; the latter are responsive to upstream
regulators quite distinct from the “stress-activated” MAP ki-
nases (7, 8). Since we have shown that Pak(s) can be activated
by mammalian G-protein-coupled receptors (15) and growth
factor receptors,
it is likely that signaling through Pak con-
tributes to the activation of stress-activated MAP kinases by
such stimuli as well (25). Based on these data, we suggest a
pathway, depicted in Fig. 4, through which a variety of up-
stream signaling molecules can stimulate activity of the p38
and JNK kinases. Activation of Rac and/or Cdc42 by upstream
signals leads to increased activity of Pak kinase(s). Pak does
not directly phosphorylate p38 or JNK1, and both p38 and JNK
are known to require phosphorylation of both Thr and Tyr
residues for activation to occur (12). This dual phosphorylation
is mediated by the action of upstream MAP kinase kinases,
which are in turn controlled by MAP kinase kinase kinases in
a typical MAP kinase regulatory cascade (21–23). We therefore
suggest it is likely that, by analogy with the Ste20 kinase
cascade in S. cerevisiae, Paks regulate the activity of MAP
kinase kinase kinases, which act in turn on MAP kinase ki-
nases to directly phosphorylate and regulate p38 and JNK.
Potentially, Paks may serve to coordinate stress responses at
the transcriptional level with morphological and cytoskeletal
changes that occur concomitantly. Thus, regulation of Rac and
Cdc42 function may be an important component of the mam-
malian response to shock and other inflammatory disorders.
Acknowledgments—We thank Yan Wang for assistance in tissue
culture and Toni Lestelle for excellent secretarial support.
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FIG.4.Proposed signal transduction pathway for activation of
the stress-regulated p38 and JNK MAP kinases. MKK, MAP ki-
nase kinase; MKKK, MAP kinase kinase kinase.
p21-activated Kinases Regulate p38 Activity23936
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Ulevitch and Gary M. Bokoch
Jonathan Chernoff, Ulla G. Knaus, Richard J.
Shengjia Zhang, Jiahuai Han, Mary Ann Sells,
the Downstream Mediator Pak1
Mitogen-activated Protein Kinase through
Rho Family GTPases Regulate p38
Cell Biology and Metabolism:
doi: 10.1074/jbc.270.41.23934
1995, 270:23934-23936.J. Biol. Chem. the most updated version of this article at
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... Both Arp2/3-and mDia2-nucleated filaments can be considered as "building blocks" for contractile SFs [32]. Located downstream of Rac1 and Cdc42, activated/phosphorylated F-actin side-binding heat shock protein 27 (HSP-27) also supports actin polymerization by dissociating from F-actin [102][103][104][105]. HSP-27 is phosphorylated by MAPKAPK2, which (i) is part of the p38 MAPK pathway downstream of Rac1 and Cdc42 [106]; and (ii) phosphorylates/activates LIMK1 [107] and p16-Arc, a part of the Arp2/3 complex [108,109]. Moreover, Rac1 induces the activation of mitogen-activated protein kinase kinase (MKK) 3 and MKK6 for activation of p38 MAPK [110][111][112] to phosphorylate HSP-27 for supporting actin polymerization. ...
... Moreover, Rac1 induces the activation of mitogen-activated protein kinase kinase (MKK) 3 and MKK6 for activation of p38 MAPK [110][111][112] to phosphorylate HSP-27 for supporting actin polymerization. Interestingly, vascular endothelial growth factor (VEGF) also induces actin polymerization through the p38-MAPKAPK2-LIMK-cofilin [107] and p38-MAPKAPK2-HSP-27 pathways [104] and signals upstream of p38 through MKK3 [110] and PAK1 [106], which is activated by Cdc42 [106,113] and Rac [106,114]. ...
... Moreover, Rac1 induces the activation of mitogen-activated protein kinase kinase (MKK) 3 and MKK6 for activation of p38 MAPK [110][111][112] to phosphorylate HSP-27 for supporting actin polymerization. Interestingly, vascular endothelial growth factor (VEGF) also induces actin polymerization through the p38-MAPKAPK2-LIMK-cofilin [107] and p38-MAPKAPK2-HSP-27 pathways [104] and signals upstream of p38 through MKK3 [110] and PAK1 [106], which is activated by Cdc42 [106,113] and Rac [106,114]. ...
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... The FOS gene encodes the proto-oncogene c-FOS protein and NFATs, which are known widely for their cytokine gene expression properties and have been increasingly shown to regulate other genes related to cell cycle progression, cell differentiation, and apoptosis [39,40]. Late phase, SEB p.e. up-regulated genes that encode oncoproteins, such as Rho GTPase, which is also linked with ERK/MAPK [41]. Consequently, the G2/M DNA Damage Checkpoint Regulation, a critical biofunction closely linked with apoptosis, was highly perturbed. ...
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Staphylococcus aureus, a gram-positive bacterium, causes toxic shock through the production of superantigenic toxins (sAgs) known as Staphylococcal enterotoxins (SE), serotypes A-J (SEA, SEB, etc.), and toxic shock syndrome toxin-1 (TSST-1). The chronology of host transcriptomic events that characterizes the response to the pathogenesis of superantigenic toxicity remains uncertain. The focus of this study was to elucidate time-resolved host responses to three toxins of the superantigenic family, namely SEA, SEB, and TSST-1. Due to the evolving critical role of melanocytes in the host’s immune response against environmental harmful elements, we investigated herein the transcriptomic responses of melanocytes after treatment with 200 ng/mL of SEA, SEB, or TSST-1 for 0.5, 2, 6, 12, 24, or 48 h. Functional analysis indicated that each of these three toxins induced a specific transcriptional pattern. In particular, the time-resolved transcriptional modulations due to SEB exposure were very distinct from those induced by SEA and TSST-1. The three superantigens share some similarities in the mechanisms underlying apoptosis, innate immunity, and other biological processes. Superantigen-specific signatures were determined for the functional dynamics related to necrosis, cytokine production, and acute-phase response. These differentially regulated networks can be targeted for therapeutic intervention and marked as the distinguishing factors for the three sAgs.
... p21-activated kinases (PAKs) are members of a family of serine/ threonine protein kinases that are central to cellular regulation and signal transduction (Hofmann, 2004;Kumar et al., 2006;Manser et al., 1994). PAKs regulate a wide range of cellular functions, including cytoskeletal actin assembly, neurite outgrowth, cell migration, apoptosis, cell cycle, survival, gene transcription, hormone signaling, and mitogen-activated protein kinase (MAPK) pathways (Bokoch, 2003;D rijard, 1995;Rudel, 1997;Sells et al., 1999;Zhang et al., 1995). In mammals, six PAK isoforms have been identified, and they are classified into two groups, namely group I and group II, based on their domain architecture and regulatory mechanisms. ...
The p21-activated kinases (PAKs) belong to serine/threonine kinases family, regulated by ∼21 kDa small signaling G proteins RAC1 and CDC42. The mammalian PAK family comprises six members (PAK1–6) that are classified into two groups (I and II) based on their domain architecture and regulatory mechanisms. PAKs are implicated in a wide range of cellular functions. PAK1 has recently attracted increasing attention owing to its involvement in oncogenesis, tumor progression, and metastasis as well as several life-limiting diseases and pathological conditions. In Caenorhabditis elegans, PAK1 functions limit the lifespan under basal conditions by inhibiting forkhead transcription factor DAF-16. Interestingly, PAK depletion extended longevity and attenuated the onset of age-related phenotypes in a premature-aging mouse model and delayed senescence in mammalian fibroblasts. These observations implicate PAKs as not only oncogenic but also aging kinases. Therefore, PAK-targeting genetic and/or pharmacological interventions, particularly PAK1-targeting, could be a viable strategy for developing cancer therapies with relatively no side effects and promoting healthy longevity. This review describes PAK family proteins, their biological functions, and their role in regulating aging and longevity using C. elegans. Moreover, we discuss the effect of small-molecule PAK1 inhibitors on the lifespan and healthspan of C. elegans.
... PAK proteins mediate many effects including dissolution of stress fibres and reorganisation of focal complexes in HeLa cells (Manser et al., 1997;Zhao et al., 1998), activate the JNK pathway but whether this is in a Cdc42/Rac-dependent or independent manner is a point of discussion (Zhang et al., 1995;Teramoto H et al., 1996b;Westwick et al., 1997). PAK is required for Ras-mediated transformation in Rat-1 fibroblasts (Tang Y et al., 1998). ...
Rac is a member of the Rho family of low molecular weight GTPases (p21s) which is involved in diverse processes including regulation of the actin cytoskeleton and transcriptional activation. Chimaerin, a multidomain GTPase activating protein (GAP) downregulates Rac by increasing its intrinsic rate of GTP hydrolysis. Two splice variants of the chimaerin gene differ in tissue and developmental expression patterns and α2-chimaerin contains an N terminal SH2 domain which is absent from αl-chimaerin. The distribution and morphological effects of the chimaerins, α2-chimaerin SH2 domain mutants and potential α2-chimaerin targets in NIE 115 neuroblastoma cells were investigated. The distribution of α1-chimaerin was predominantly cytoskeletal and α2-chimaerin cytosolic. In transiently transfected NIE 115 cells, α1-chimaerin was concentrated in the perinuclear region and its expression induced cell rounding, whilst α2-chimaerin was expressed throughout flattened, neurite bearing cells. A point mutation in the SH2 domain of α2-chimaerin induced an α1-chimaerin-like protein distribution and morphology. The effects of long term chimaerin overexpression on cell morphology and potential protein interactions were also investigated. Overexpression of α2-chimaerin induced an enlarged, flattened morphology and neurite outgrowth in the presence of serum, whilst overexpression of α1-chimaerin induced a rounded morphology with multiple peripheral actin microspikes and inhibited neurite outgrowth. p35, the neuronal cdk5 regulator and also an 130 kDa tyrosine phosphorylated protein were immunoprecipitated with chimaerin from these cell lines. Similarly an 180 kDa tyrosine phosphorylated protein was identified as a potential target of the α2-chimaerin SH2 domain. Investigation into the effects of chimaerin on activation of the transcription factor NFKB demonstrated cell type specific differences in NFKB signalling pathways between HeLa and NIE 115 cells. These results suggest that functional differences in the chimaerin isoforms are specified by the divergent N terminal sequences.
... ERK activation was found to be required for H-Ras-mediated migration and invasiveness of human breast epithelial MCF10A cells [11]. Previous studies have shown that p38 lies downstream of the Ras-related GTP-binding proteins Rac and Cdc42, and is directly activated by MKK3, MKK6, and MKK4 [30][31][32][33]. It appears that the p38-and ERK-mediated signaling pathways are independent of each other, and both pathways cooperate in H-Ras-mediated migrative and invasive responses in MCF10A cells. ...
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Aberrant activation of Ras has been implicated in aggressiveness of breast cancer. Among Ras isoforms (H-, K-, and N-), H-Ras has been known to be primarily responsible for invasion and metastasis of breast cancer cells. Phosphorylation of serine (Ser) or threonine (Thr) is a key regulatory mechanism responsible for controlling activities and functions of various proteins involved in intracellular signal transduction. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, Pin1 changes the conformation of a subset of proteins phosphorylated on Ser/Thr that precedes proline (Pro). In this study we have found that Pin1 is highly overexpressed in human breast tumor tissues and H-Ras transformed human mammary epithelial (H-Ras MCF10A) and MDA-MB-231 breast cancer cells. Notably, Pin1 directly bound to the activated form of H-Ras harbouring a Ser/Thr-Pro motif. Pharmacologic inhibition of Pin1 reduced clonogenicity of MDA-MB-231 human breast cancer cells. Paclitaxel accelerates apoptosis in Pin1 silenced H-Ras MCF10A cells. MDR genes (MDR1 and MRP4) were significantly downregulated in MDA-MB-231 cells stably silenced for Pin1. We speculate that Pin1 interacts with GTP-H-Ras, thereby upregulating the expression of drug resistance genes, which confers survival advantage and aggressiveness of breast cancer cells under chemotherapy.
... Microtubule involvement is consistent with the pioneering studies by George Cooper (4th) who showed that p21activated kinase-1 (Pak1)-dependent microtubule assembly plays a central role in the early response to pressure overload and mechanical stretch in right ventricular cardiomyocytes (Cheng et al., 2012a). Indeed, Pak1 regulates exercise-induced cardiac hypertrophy (Davis et al., 2015), which aligns nicely with our 2 day atrial analyses showing exercise-induced upregulation of Flna (filamen A), a cytoprotective protein that is upregulated with mechanical stress (D'Addario et al., 2003) and is essential for actin/cytoskeletal dynamics (Vadlamudi et al., 2002) through interdependent p38- (D'Addario et al., 2002) and Pak1-mediated signaling (Zhang et al., 1995;Shifrin et al., 2012). We also found enrichment of other pathways, including IQGAPs and AMPKs, in acutely exercised WT atria which are involved in the early compensatory responses to pressure overload stimuli that may be harbingers of fibrotic remodeling in the long-term (Hermida et al., 2013;Hedman et al., 2015;Daskalopoulos et al., 2016). ...
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Atrial Fibrillation (AF) is the most common supraventricular tachyarrhythmia that is typically associated with cardiovascular disease (CVD) and poor cardiovascular health. Paradoxically, endurance athletes are also at risk for AF. While it is well-established that persistent AF is associated with atrial fibrosis, hypertrophy and inflammation, intensely exercised mice showed similar adverse atrial changes and increased AF vulnerability, which required tumor necrosis factor (TNF) signaling, even though ventricular structure and function improved. To identify some of the molecular factors underlying the chamber-specific and TNF-dependent atrial changes induced by exercise, we performed transcriptome analyses of hearts from wild-type and TNF-knockout mice following exercise for 2 days, 2 or 6 weeks of exercise. Consistent with the central role of atrial stretch arising from elevated venous pressure in AF promotion, all 3 time points were associated with differential regulation of genes in atria linked to mechanosensing (focal adhesion kinase, integrins and cell-cell communications), extracellular matrix (ECM) and TNF pathways, with TNF appearing to play a permissive, rather than causal, role in gene changes. Importantly, mechanosensing/ECM genes were only enriched, along with tubulin- and hypertrophy-related genes after 2 days of exercise while being downregulated at 2 and 6 weeks, suggesting that early reactive strain-dependent remodeling with exercise yields to compensatory adjustments. Moreover, at the later time points, there was also downregulation of both collagen genes and genes involved in collagen turnover, a pattern mirroring aging-related fibrosis. By comparison, twofold fewer genes were differentially regulated in ventricles vs. atria, independently of TNF. Our findings reveal that exercise promotes TNF-dependent atrial transcriptome remodeling of ECM/mechanosensing pathways, consistent with increased preload and atrial stretch seen with exercise. We propose that similar preload-dependent mechanisms are responsible for atrial changes and AF in both CVD patients and athletes.
p>Interleukin 10 has proven itself to be an important regulator of inflammation with a wide range of biological effects. These range from a down regulation of cytokine, chemokine and superoxide production within the macrophage, to an up regulation of immunoglobin secretion in B-cells. However, despite Interleukin 10 being an important anti-inflammatory cytokine, very little is understood about how this cytokine is regulated within the cell. During this study we investigated how Interleukin 10 is regulated in the monocyte and macrophage involved in inflammation. In the first part of the study a sandwich ELIS A was used with FACS analysis, to characterise the profile of Interleukin 10 to purified bacterial lipopolysaccaride and map their secretion to the individual cells involved in inflammation. Using pharmacological specific inhibitors the second messenger pathways were mapped. We have looked at how Interleukin 10 is regulated by the MAP kinase pathways. Using potent and selective MAP kinase inhibitors we have demonstrated in various cell types how inhibition of the p38 and JNK MAP kinase pathways led to a down regulation in Interleukin 10 production. Once the cellular pathways that regulate Interleukin 10 production were worked out, we were able to continue to work further down the regulatory pathways to reach the DNA and begin to investigate which transcription factors are important in Interleukin 10 transcription and translation. Understanding how this important cytokine is regulated can lead to the development of a novel treatment for patients who suffer fi-om tissue destructive and painful inflammation diseases.</p
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Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Importance Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.
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Cells process information via signal networks that typically involve multiple components which are interconnected by feedback loops. The combination of acute optogenetic perturbations and microscopy-based fluorescent response readouts enables the direct investigation of causal links in such networks. However, due to overlaps in spectra of photosensitive and fluorescent proteins, current approaches that combine these methods are limited. Here, we present an improved chemo-optogenetic approach that is based on switch-like perturbations induced by a single, local pulse of UV light. We show that this approach can be combined with parallel monitoring of multiple fluorescent readouts to directly uncover relations between signal network components. We present the application of this technique to directly investigate feedback-controlled regulation in the cell contraction signal network that includes GEF-H1, Rho and Myosin, and functional interactions of this network with tumor relevant RhoA G17 mutants.
The actin cytoskeleton is crucial for oligodendrocyte differentiation and myelination. Here we show that p21-activated kinase1 (PAK1), a well-known actin regulator, promotes oligodendrocyte morphological change and myelin production in the central nervous system. A combination of in vitro and in vivo models demonstrated that PAK1 is expressed throughout the oligodendrocyte lineage with highest expression in differentiated oligodendrocytes. Inhibiting PAK1 early in oligodendrocyte development decreased oligodendrocyte morphological complexity and altered F-actin spreading at the tips of oligodendrocyte progenitor cell processes. Constitutively activating AKT in oligodendrocytes in male and female mice, which leads to excessive myelin wrapping, increased PAK1 expression, suggesting an impact of PAK1 during active myelin wrapping. Furthermore, constitutively activating PAK1 in oligodendrocytes in zebrafish led to an increase in myelin internode length while inhibiting PAK1 during active myelination decreased internode length. As myelin parameters influence conduction velocity, these data suggest that PAK1 may influence communication within the CNS. These data support a model in which PAK1 is a positive regulator of CNS myelination.SIGNIFICANCE STATEMENT Myelin is a critical component of the central nervous system (CNS) that provides metabolic support to neurons and also facilitates communication between cells in the CNS. Recent data demonstrate that actin dynamics drives myelin wrapping but how actin is regulated during myelin wrapping is unknown. The authors investigate the role of the cytoskeletal modulator PAK1 during differentiation and myelination by oligodendrocytes, the myelinating cells of the CNS. They demonstrate that PAK1 promotes oligodendrocyte differentiation and myelination by modulating myelin internode length, thus playing a critical role in the function of the CNS.
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Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.
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Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.
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We report on the construction and use of two eukaryotic expression vectors which add well-characterized epitope tags to the N termini of proteins. The utility of these vectors is demonstrated for detecting the expression of a variety of proteins. As the addition of these epitope tags can in some cases obviate the need to generate specific antisera to each individual protein, these vectors provide a facile means both to monitor protein expression and to purify such expressed proteins.
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The conversion of cholesterol into bile acids in the liver represents the major catabolic pathway for the removal of cholesterol from the body. In this complex biosynthetic pathway, at least 10 enzymes modify both the ring structure and side chain of cholesterol, resulting in the formation of the primary bile acids, cholic acid, and chenodeoxycholic acid. To gain insight into the details and regulation of this pathway, we have used protein sequencing and molecular cloning techniques to isolate and characterize a cDNA encoding the rabbit mitochondrial sterol 26-hydroxylase. This enzyme catalyzes the first step in the oxidation of the side chain of sterol intermediates in the biosynthesis of bile acids. The structure of the sterol 26-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, reveals it to be a mitochondrial cytochrome P-450. A signal sequence of 36 residues precedes a coding region of 499 amino acids, predicting a molecular weight of 56,657 for the mature protein. The identity of the 26-hydroxylase cDNA was further confirmed by expression in monkey COS cells employing a versatile eukaryotic expression vector. Blotting experiments revealed that the mRNA for this enzyme is expressed in many tissues and that it is encoded by a low copy number gene in the rabbit genome.
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In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described, not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization of these newly identified family members.
Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.
Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.
Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.
The c-fos serum response element (SRE) forms a ternary complex with the transcription factors SRF (serum response factor) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38. Functional Rho is required for regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway.
The Rho subfamily of GTPases is involved in control of cell morphology in mammals and yeast. The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA did not exhibit this activity, and none of the proteins activated the ERK subgroup of MAPKs. JNK, but not ERK, activation was also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation.