Rac GTPase signaling through the PP5
Saverio Gentile*, Thomas Darden*, Christian Erxleben*, Charles Romeo*, Angela Russo*, Negin Martin*,
Sandra Rossie†, and David L. Armstrong*‡
*Environmental Biology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human
Services, Research Triangle Park, NC 27709; and†Department of Biochemistry and Purdue Cancer Center, Purdue University, West Lafayette, IN 47907
Communicated by Lutz Birnbaumer, National Institutes of Health, Research Triangle Park, NC, January 4, 2006 (received for review September 20, 2005)
We have investigated the Rac-dependent mechanism of KCNH2
channel stimulation by thyroid hormone in a rat pituitary cell line,
GH4C1, with the patch-clamp technique. Here we present physio-
logical evidence for the protein serine?threonine phosphatase,
PP5, as an effector of Rac GTPase signaling. We also propose and
Inhibition of PP5 with the microbial toxin, okadaic acid, blocked
channel stimulation by thyroid hormone and by Rac, but signaling
was restored by expression of a toxin-insensitive mutant of PP5,
Y451A, which we engineered. PP5 is unique among protein phos-
phatases in that it contains an N-terminal regulatory domain with
three tetratricopeptide repeats (TPR) that inhibit its activity. Ex-
pression of the TPR domain coupled to GFP blocked channel
stimulation by the thyroid hormone. We also show that the
published structures of the PP5 TPR domain and the TPR domain of
p67, the Rac-binding subunit of NADPH oxidase, superimpose over
92 ? carbons. Mutation of the PP5 TPR domain at two predicted
contact points with Rac-GTP prevents the TPR domain from func-
tioning as a dominant negative and blocks the ability of Y451A to
rescue signaling in the presence of okadaic acid. PP5 stimulation by
Rac provides a unique molecular mechanism for the antagonism of
Rho-dependent signaling through protein kinases in many cellular
processes, including metastasis, immune cell chemotaxis, and neu-
KCNH2 ? tetratricopeptide repeat ? neuronal development ? potassium
channel ? thyroid hormone
of the actin cytoskeleton (1), but they also participate in many
other important cellular processes (2), such as the innate im-
mune response that stimulates superoxide production in neu-
trophils through Rac-dependent activation of the NADPH ox-
idase (3). In many of these other processes, Rac and Rho
generally produce antagonistic effects, particularly in the devel-
oping nervous system, where they have opposite effects on
neuronal polarity, neurite outgrowth, and synaptic plasticity (4).
Although Rho signals primarily through several protein serine?
threonine (S?T) kinases (5), none of the Rac effectors identified
thus far provide a simple explanation for the functional antag-
onism between Rac and Rho.
We reported previously that Rac and Rho mediate the op-
posing effects of the thyroid hormone, 3,5,3?-triiodothyronine
(T3), and thyrotropin-releasing hormone (TRH) on KCNH2
channel activity in pituitary cells (6), but the mechanism of
channel regulation by Rac or Rho was not determined. The
conclusions from that study are summarized in Fig. 1A. KCNH2
proteins are voltage-activated potassium channels that regulate
spike frequency in electrically excitable cells, such as cardiac
myocytes and the endocrine cells of the pituitary, through their
unique kinetics of inactivation and recovery (7). KCNH2 chan-
nels are known to be phosphorylated and inhibited by the
cAMP-dependent protein kinase (8), and recovery from inhibi-
tion by TRH requires protein phosphatase activity (9). Conse-
ac and Rho are members of the same family of Ras-related
quently, we postulated that Rho inhibits the channels via a
protein S?T kinase, whereas Rac stimulates the channels via a
protein S?T phosphatase. Here we demonstrate that the Rac-
dependent effects of thyroid hormone on KCNH2 potassium
channel activity in a rat pituitary cell line (6) are mediated by the
protein S?T phosphatase, PP5 (10), which is expressed in all
tissues, including the brain (11, 12). We also propose and test a
structural model for direct PP5 activation by Rac-GTP, which
involves residues conserved in all three Rac isoforms, but not in
the Rho or Cdc42 family members. PP5 is unique among protein
phosphatases in that it contains three tetratricopeptide repeats
(TPRs) (13), which inhibit its activity (14, 15). We show that the
published structures of the PP5 TPR domain (13) and the first
three TPRs of p67, the Rac-binding subunit of NADPH oxidase
(16), can be superimposed over 92 ? carbons. Mutation of PP5
at two predicted contact points with Rac-GTP blocks channel
stimulation by thyroid hormone. Rac-dependent stimulation of
a protein S?T phosphatase provides a general mechanism for the
antagonism between Rac and Rho.
KCNH2 current amplitude was measured under voltage-clamp
through gramicidin-perforated patches on GH4C1cells, as de-
scribed in the Materials and Methods. The unique kinetics of
KCNH2 channels (7), which inactivate rapidly after activation at
positive voltages and then transiently reactivate during repolar-
ization, allow one to separate the potassium current through
KCNH2 channels from other potassium currents with the volt-
age protocol shown in Fig. 1B. Representative currents are
shown in Fig. 1C. Note that the class III antiarrhythmic drug
E-4031, which selectively inhibits KCNH2 channels (17), com-
pletely blocks the KCNH2 currents recorded during the negative
step to ?120 mV. T3 rapidly increases the amplitude of the
E-4031-sensitive KCNH2 current by ?50%, and this response is
sustained in metabolically intact cells voltage-clamped through
perforated patches (Fig. 1D).
To test the involvement of protein S?T phosphatases in
Rac-dependent stimulation of KCNH2 channels, we exposed the
cells to okadaic acid, a dinoflagellate toxin that is concentrated
by shellfish during algal blooms. At a concentration of 100 nM,
okadaic acid completely inhibits several protein S?T phospha-
tases, including PP2A, PP4, and PP5, but not other protein S?T
phosphatases such as PP1 or PP2B (18). Bath application of 100
nM okadaic acid for 5 min had no significant effect on the
amplitude of the basal, E-4031-sensitive current. However, oka-
daic acid completely prevented the increase in KCNH2 current
in response to a subsequent application of T3 (Fig. 1E). In
contrast, 1 ?M fostriecin, which inhibits PP2A and PP4 but not
Conflict of interest statement: No conflicts declared.
Abbreviations: S?T, serine?threonine; T3, 3,5,3?-triiodothyronine; TPR, tetratricopeptide
repeat; HA, hemagglutinin.
‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
March 28, 2006 ?
vol. 103 ?
no. 13 www.pnas.org?cgi?doi?10.1073?pnas.0600080103
PP5 (18), had no effect on channel stimulation by T3. In both
control conditions and the presence of fostriecin, peak current
was increased 1.7-fold by T3(Fig. 1E). Because PP5 is the only
S?T phosphatase that is inhibited completely by 100 nM okadaic
acid but not at all by 1 ?M fostriecin, we investigated whether
PP5 is required for Rac-dependent stimulation of KCNH2
channels by T3.
To provide a more direct test for PP5 involvement in channel
stimulation by T3, we engineered a mutant form of PP5 with
decreased sensitivity to inhibition by okadaic acid. Structural
studies have identified the toxin-binding site in the C terminus
of PP1 (19), and this sequence is conserved in all of the protein
S?T phosphatases inhibited by okadaic acid, including PP5 (Fig.
2A). Y272 in PP1 was reported to be particularly important for
of its lipid-stimulated enzymatic activity to inhibition by okadaic
inhibition of the flag-tagged PP5 is ?30 nM, but for the Y451A
mutant, it is ?10 ?M (Fig. 2B). In cells transfected with the
Y451A mutant of PP5, T3increased the KCNH2 current am-
plitude by ?50% in the presence of 100 nM okadaic acid (Fig.
2C). This result demonstrates that PP5 alone is sufficient to
restore KCNH2 stimulation in the presence of okadaic acid,
strongly supporting the involvement of PP5 in T3 signaling.
Furthermore, PP5 is required downstream of Rac in KCNH2
stimulation by T3, because okadaic acid also prevented the
stimulation of KCNH2 currents produced by dialyzing the
GH4C1cells with the constitutively active Rac1 Q61L protein
PP5 is unique among protein phosphatases in that it contains
three TPRs in the amino terminus (13), which inhibit its
phosphatase activity (14, 15). The TPR is a degenerate 34-aa
motif that forms two antiparallel helices (21). TPRs are ex-
pressed in a wide variety of proteins, usually as arrays of 3–10
tandem repeats, and typically mediate binding to protein part-
ners (21). A well documented effector of Rac, the p67 subunit
of the NADPH oxidase, binds Rac-GTP through its own TPR
domain (16). Using the O program (22), we superimposed the
published structures of the three TPRs of PP5 (13) onto the first
three TPRs of p67 (16). The two structures superimpose very
closely, with a rms error equal to 0.1 nm over 92 ? carbons (Fig.
3A). We confirmed that PP5 associates with Rac by coexpressing
hemagglutinin (HA)-tagged Rac1 Q61L and Flag-PP5 in GH4C1
cells. Flag-PP5 was immunoprecipitated together with HA-Rac
by the anti-HA antibody, but not by control IgG (Fig. 3B).
To test the role of the TPR domain in Rac-dependent
stimulation of PP5, we constructed a chimera containing resi-
dues 1–179 of PP5, which encompasses the three TPRs but lacks
the catalytic domain, which we replaced with GFP (Fig. 3C). We
reasoned that this construct might function as a dominant
negative by competing with native PP5 for protein partners, as
others have demonstrated (23). In support of our hypothesis,
immunoprecipitation of Rac1 Q61L coprecipitated TPR-GFP
from GH4C1 cells transiently expressing both constructs (Fig.
3B). More importantly, transient expression of the PP5 TPR-
GFP construct in GH4C1 cells completely blocked KCNH2
stimulation by T3(Fig. 3C). In contrast, the TPR-GFP construct
had no effect on the lipid-stimulated phosphatase activity of
wild-type PP5 in vitro (not shown), and KCNH2 stimulation by
T3in cells transfected with GFP alone was normal (not shown).
Because the TPR-GFP construct blocked T3signaling in GH4C1
cells but not PP5 activity in vitro, we conclude that a protein that
binds to TPR domains is required for T3 signaling. The Rac
GTPase fits both these criteria. It is required for T3signaling (6)
and is known to bind to a similar TPR domain in p67 (16).
Although PP5 is the only protein S?T phosphatase with an
identified TPR domain, many other proteins in mammalian cells
contain TPR domains (21), so we investigated the specificity of
this effect. Based on the similarity between the secondary
structures of the TPR domains in PP5 and p67 (Fig. 3A), we
acid. (A) Diagrammatic summary of ref. 6. KCNH2 stimulation by thyroid
hormone (T3) was shown to be Rac-dependent and opposed by Rho. (B)
Voltage protocol for isolating current through KCNH2 channels. (C) KCNH2
current at ?120 mV is increased by 100 nM T3and blocked completely by 10
?M E-4031, a selective KCNH2 antagonist. (D) Time course of changes in
KCNH2 current amplitude obtained from metabolically intact cells voltage-
clamped through gramicidin-perforated patches. (E) Peak amplitude histo-
grams of the E-4031-sensitive current before and 3 min after bath application
of 100 nM of the T3hormone in control cells and in cells exposed to 100 nM
okadaic acid or 1 ?M fostriecin for 5 min before T3application.
Gentile et al. PNAS ?
March 28, 2006 ?
vol. 103 ?
no. 13 ?
of p67 in the complex with Rac1-GTP (Fig. 4A). In this model,
the exposed switch 1 loop of activated Rac-GTP binds to the
TPR domain of PP5 on the side opposite the TPR interaction
with the PP5 active site (24). If the interaction with Rac pulled
the TPR domain out of the active site, it would provide a
plausible mechanism for PP5 stimulation by activated Rac. The
model also allowed us to identify K126 in rat PP5, corresponding
bonding simultaneously with the side chain oxygens of three
different Rac residues, S22, N26, and Q162 (Fig. 4A). We also
identified an important contact between D92?K93 in PP5, which
correspond to D67?K68 in p67, with the backbone amine and
carbonyl of Rac G30 in switch 1. Rac G30 is the only residue
contacting p67 that is not conserved in any other Rho family
members (Fig. 4B). When we substituted both K93E and K126A
in the PP5 TPR-GFP construct, it no longer inhibited KCNH2
channel stimulation (Fig. 4E). Furthermore, when either K93E
or K126A was substituted in PP5 Y451A, the construct lost its
ability to rescue T3signaling in the presence of okadaic acid (Fig.
Schematic diagram showing the domain structure of the protein Ser?Thr
phosphatases PP5 and PP1. Blue and orange boxes indicate the TPR and the
catalytic domains, respectively. The C-terminal phosphatase sequences impli-
cated in toxin binding are shown. (B) Enzymatic activity of recombinant PP5
constructs stimulated by arachidonic acid in the presence of okadaic acid.
Open circles, wild-type PP5; closed squares, Flag-tagged PP5 mutated at
Y451A; closed circles, Flag-tagged wild-type PP5. (C) In cells transfected with
PP5 Y451A, T3stimulates KCNH2 channels in the presence of 100 nM okadaic
acid. (D) In cells dialyzed with Rac1 Q61L, the basal currents are larger but
pretreatment with 100 nM okadaic acid prevented the increase.
PP5 Y451A rescues T3signaling in the presence of okadaic acid. (A)
showing the superposition of the three TPR motifs of PP5 (blue) with the first
error of 0.1 nm over 92 ? carbons. (B) Immunoprecipitation of HA-tagged
constitutively active Rac Q61L in lysates from GH4C1 cells that had been
mouse IgG from cells transfected with Rac1 Q61L and Flag-PP5 (lane 4) failed
to coprecipitate PP5. (C) In TPR-GFP expressing cells, basal KCNH2 currents are
normal, but stimulation by T3is blocked.
TPR of PP5 mediate Rac-dependent signaling. (A) Ribbon diagram
www.pnas.org?cgi?doi?10.1073?pnas.0600080103Gentile et al.
4 C and D), despite being expressed normally (not shown). Thus,
interaction with the TPR domain appears to be critical for Rac
signaling through PP5.
Together, these results provide structural and physiological
evidence for PP5 as a direct molecular effector for activated
Rac-GTP. Rho-GTP has been shown to inhibit myosin phos-
phatase activity in smooth muscle, but this appears to be an
indirect mechanism mediated by Rho kinase phosphorylation of
a PP1 inhibitor protein (25). The heterotrimeric G proteins,
G?12and G?13, have been reported to stimulate PP5 in vitro (26),
but neither the structural mechanism nor the physiological
significance of this effect has been established. G?13is unlikely
to stimulate PP5 in GH4C1cells because, unlike T3and Rac,
which require PP5 to stimulate KCNH2 channels (Fig. 2),
constitutively active G13reduces KCNH2 current amplitude (6).
The mechanism we propose is not unique. It is based on the well
documented stimulation of NADPH oxidase by Rac through
binding to the TPR domain of p67 (3, 16). Despite the very
are essentially identical (Fig. 3A). Thus, other proteins with TPR
domains might now be investigated for regulation by Rac.
The evidence presented here also identifies Rac as a hormon-
ally regulated activator for PP5. We initially identified PP5 as a
lipid-stimulated protein phosphatase in brain extracts (11),
having postulated that pertussis toxin-sensitive heterotrimeric G
proteins might increase phosphatase activity by stimulating
phospholipase A2 to produce arachidonic acid (27). However,
receptor activation of G?i and G?o also stimulates the activity
of the ? isoform of phosphatidylinositol 3 kinase through G??
subunits (28), and phosphatidylinositol 3,4,5 tris phosphate
(PIP3) is known to activate guanine exchange factors for Rac
(29). In addition, we have obtained separate evidence that
channel stimulation by thyroid hormone is mediated by the
nuclear receptor for thyroid hormone, TR?, stimulating phos-
phatidylinositol 3 kinase (30). Phosphatidylinositol 3 kinase has
of other nuclear hormones (31). Thus, Rac-dependent stimula-
tion of PP5 could account for many other examples of ion
channel regulation by nuclear and G protein-coupled receptors.
Finally, although Rho and Rac signaling pathways interact at
many levels (2), Rac-dependent stimulation of PP5 provides a
direct molecular mechanism for the antagonism of Rho-
dependent signaling through protein kinases in many fundamen-
tal cellular processes linked to human disease, including cancer
(32), inflammation (3), and mental retardation (33). Rac-
dependent stimulation of PP5 also provides a molecular mech-
anism for antagonism of apoptosis (34) in degenerative diseases
of the aging (35–37) nervous system.
Materials and Methods
GH4C1cells were grown in DMEM?F12 with 10% calf serum?50
mg/liter streptomycin?31 mg/liter penicillin?640 mg/liter
NaHCO3. Cells were plated on glass coverslips (Deutsche
Lipofectamine 2000 (Invitrogen) with the PP5 and Rac plasmids
together with a separate plasmid encoding GFP at a ratio of 10:1.
Fluorescent cells were used for recordings between 12 and 48 h
Recombinant PP5 Constructs. The reference sequence for PP5
cloning was the rat ‘‘ppt’’ (RNPPT) sequence (GenBank acces-
sion no. X77237). Rat PP5 was cloned by PCR as a MluI-NotI
fragment and inserted into the pCI vector engineered to contain
a Flag epitope at the N terminus. The TPR-GFP chimera was
created by cutting the Flag-PP5 construct at amino acid 179 with
HindIII and NotI and substituting GFP (Clontech) by using the
following two oligos to PCR a GFP that could be fused to the
TPR domain at the HindIII site: cgc ggg aag ctt gcc ATG GTG
AGC AAG GGC GAG GAA CTG TTC (the HindIII site is
underlined, uppercase letters are a GFP sequence from CLON-
TECH); CAT GGC ATG GAC CTG TAC AAG TAA agc ggc
cgc ccc gcg (the NotI site underlined). Single mutations in PP5
diagram of a model for the PP5-TPR (blue) complex with Rac1-Q61L-GTP
(green). GTP is shown as sticks with carbon atoms in green, oxygen atoms in
red, and phosphates in magenta. (Inset) The hydrogen bonds between PP5
K126 and the sidechain oxygens of Rac S22, N26, and Q162, and between PP5
K93 and the backbone carbonyl of Rac G30. (B) Sequence alignment of the
nor Cdc42, have all four of the critical residues (highlighted in red) that are
proposed to contact PP5. (C and D) Peak amplitude histograms of the KCNH2
current in 100 nM okadaic acid from control cells or from cells expressing
application of 100 nM of the T3hormone in cells expressing the TPR domain
of PP5 with the mutations K93E and K126A.
A structural model for PP5 stimulation by Rac-GTP. (A) Ribbon
Gentile et al. PNAS ?
March 28, 2006 ?
vol. 103 ?
no. 13 ?
were introduced by PCR with QuickChange XL site-directed Download full-text
mutagenesis kit (Stratagene). Mutant constructs were se-
quenced to confirm the presence of the desired mutations and
the absence of additional mutations.
Biochemical Assay of PP5 Activity. Recombinant PP5, Flag-PP5,
and Flag-PP5(Y451A) were expressed as GST-fusion proteins,
purified, and assayed by using32P-casein as substrate as de-
scribed (11). Flag-PP5 and Flag-PP5(Y451A) exhibited specific
activities in the range of 10–40 nmol Pireleased per min per mg
of phosphatase protein, which is similar to the activity of
wild-type recombinant PP5.
Electrophysiological Recordings and Analysis. Potassium currents
were recorded at room temperature (20–24°C) under voltage
clamp with an EPC9 patch-clamp amplifier interface (HEKA
Electronics, Lambrecht?Pfalz, Germany) by using PULSE soft-
ware (HEKA Electronics) for data acquisition, pulse generation,
and analysis. Data are presented as mean ? SE of n experiments.
The patch pipettes were made from Corning type 7052 glass
(Garner Glass, Claremont, CA). Whole-cell currents were ob-
tained conventionally from dialyzed cells voltage-clamped
through ruptured membrane patches or from metabolically
intact cells voltage-clamped through gramicidin-perforated
patches. No leak subtraction was applied. In both cases, the
solution bathing the cells contained 140 mM KCl, 0.1 mM CaCl2,
2 mM MgCl2, 10 mM Hepes, and 10 mM glucose, pH 7.4. For
dialyzed cells, the pipette contained 140 mM KCl, 2 mM MgCl2,
10 mM Hepes, 1 mM dibromoBAPTA [BAPTA, 1,2-bis(2-
aminophenoxy)ethane-N,N,N?,N?-tetraacetate], 1 mM ATP,
and 0.1 mM GTP. For perforated patches, pipettes contained
110 mM K?-gluconate, 35 mM KCl, 2 mM MgCl2, 1 mM CaCl2,
10 mM Hepes, pH 7.4, and 100 ?g?ml gramicidin. The pulse
protocol was run a minimum of 10 times before the perfusion
control and 10 times before the addition of T3to ensure that the
currents were stable. Successful isolation was confirmed in every
experiment with the class III antiarrhythmic methane-
sulfonanilide, E-4031 (10 ?M, Biomol, Plymouth Meeting, PA).
Averaged data are presented as mean ? SE. Differences were
evaluated with Student’s t test. P ? 0.05 is indicated with an
Immunoprecipitation. GH4C1 cells were transfected with HA-
Rac-Q61L alone or in combination with either Flag-PP5 or
TPR-GFP. After 24 h, cells were washed twice with ice-cold PBS
and sonicated briefly in lysis buffer (40 mM Tris, pH 7.6?5 mM
Hepes?50 mM NaCl?1 mM EDTA?1 mM EGTA?5 mM
NaF?1% glycerol?0.1% Triton X-100), complete protease in-
hibitor mixture (Roche Diagnostics) at 4°C. The lysate was
centrifuged (4°C, 15 min, 20,000 ? g) and protein in the
Equal amounts of supernatant protein were mixed with HA
monoclonal antibody (Covance, Berkeley, CA) or mouse IgG
overnight at 4°C, followed by Protein-G-agarose for 1 h more.
Immunoprecipitates were washed four times with lysis buffer.
ferred to nitrocellulose, and PP5 detected by using a PP5
antiserum (12) and enhanced chemiluminescence (Amersham
Structure Modeling. The models were created by using the O
program (22). The proposed complex between the TPR domain
of PP5 and Rac-GTP was built by a simple docking process, by
using the experimentally determined structures of the TPR
domain of PP5 alone (ref. 13; Protein Data Bank ID code 1A17)
and of the TPR domain of P67 in complex with Rac-GTP (ref.
16; Protein Data Bank ID code 1E96). The initial model was
adjusted by small rigid rotations of PP5 together with rotamer
searches for K126, K93, and a few other PP5 residues to optimize
contacts involving these sidechains while attempting to preserve
contacts analogous to those found in the experimentally deter-
mined p67-Rac-GTP structure. Figs. 1–3 were prepared by using
We thank Angela Everhart and Erica Scappini for technical assistance
and Lutz Birnbaumer, John O’Bryan, and Fernando Ribeiro-Neto for
helpful suggestions. This work was supported by the National Institutes
of Health intramural program at the National Institute for Environmen-
tal Health Sciences and by National Institutes of Health Grant NS031221
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