A PP4 Holoenzyme Balances Physiological
and Oncogenic Nuclear Factor-Kappa B
Signaling in T Lymphocytes
Markus Brechmann,1,6Thomas Mock,1,6Dorothee Nickles,2Michael Kiessling,1Nicole Weit,3Rebecca Breuer,1
Wolfgang Mu ¨ller,1Guido Wabnitz,4Felice Frey,2Jan P. Nicolay,1,5Nina Booken,5Yvonne Samstag,4
Claus-Detlev Klemke,5Marco Herling,3Michael Boutros,2Peter H. Krammer,1and Ru ¨diger Arnold1,*
1Division of Immunogenetics, Tumor Immunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120
2Division of Signaling and Functional Genomics, Department of Cell and Molecular Biology, Medical Faculty Mannheim, German Cancer
Research Center (DKFZ) and Heidelberg University, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
3Laboratory for Lymphocyte Signaling and Oncoproteome, Department of Internal Medicine, University Hospital Cologne,
50937 Cologne, Germany
4Institute for Immunology, Ruprecht-Karls-University, 69120 Heidelberg, Germany
5Department of Dermatology, Venerology and Allergology, University Medical Centre Mannheim, Ruprecht-Karls-University of Heidelberg,
68167 Mannheim, Germany
6These authors contributed equally to this work
Signal transduction to nuclear factor-kappa B
(NF-kB) involves multiple kinases and phosphory-
lated target proteins, but little is known about
signal termination by dephosphorylation. By RNAi
screening, we have identified protein phosphatase
4 regulatory subunit 1 (PP4R1) as a negative regu-
lator of NF-kB activity in T lymphocytes. PP4R1
formed part of a distinct PP4 holoenzyme and
bridged the inhibitor of NF-kB kinase (IKK) complex
and the phosphatase PP4c, thereby directing PP4c
activity to dephosphorylate and inactivate the IKK
complex. PP4R1 expression was triggered upon
activation and proliferation of primary human T
lymphocytes and deficiency for PP4R1 caused
sustained and increased IKK activity, T cell hyperac-
tivation, and aberrant NF-kB signaling in NF-kB-
addicted T cell lymphomas. Collectively, our results
unravel PP4R1 as a previously unknown activation-
associated negative regulator of IKK activity in
genic NF-kB signaling in a subgroup of T cell
Signaling to nuclear factor-kappa B (NF-kB) is crucial for T
lymphocyte activation, differentiation, and proliferation by regu-
lating a wide variety of target genes such as different cytokines
(e.g., interleukin-2 [IL-2], interferon gamma [IFN-g], and tumor
necrosis factor alpha [TNF-a]), chemokines (e.g., IL-8), and anti-
apoptotic molecules (e.g., Bcl-2, c-IAPs) (Bonizzi and Karin,
2004; Ghosh and Karin, 2002; Ruland and Mak, 2003a). NF-kB
activation in response to T cell receptor (TCR) or TNF receptor
1 (TNFR1) triggering is mediated by a high molecular weight
IkB kinase (IKK) complex (Ha ¨cker and Karin, 2006; Hayden and
Ghosh, 2004). The IKK complex comprises two enzymatic
subunits, IKKa and IKKb, and the regulatory subunit IKKg
(NEMO) (Ha ¨cker and Karin, 2006). IKK activation results in phos-
NF-kB transcription factors and render them inactive within the
cytoplasm. Phosphorylated IkB proteins are rapidly ubiquitiny-
lated and targeted for proteasomal degradation. In turn, liber-
ated NF-kB proteins, like RelA (p65), translocate into the
nucleus, where they bind to and transactivate kB sites within
the promoter region of NF-kB-regulated genes (Hayden and
Ghosh, 2008; Vallabhapurapu and Karin, 2009).
IKK activation is a transient event that is subject to several
postinductive negative feedback mechanisms ensuring proper
signal shaping, control, and termination (Acuto et al., 2008).
Prominent examples are the ubiquitin-editing enzymes A20
and the cylindromatosis tumor suppressor (CYLD), which act
as negative regulatory control elements of NF-kB. Indeed,
defects in negative regulation by A20 in lymphocytes cause
enhanced and prolonged IKK phosphorylation, resulting in hy-
peractivation of cells and promoting lymphoid malignancies
(Du ¨wel et al., 2009; Hymowitz and Wertz, 2010).
The NF-kB activating signal cascade in T lymphocytes
phosphoproteome (Ha ¨cker and Karin, 2006; Weil and Israe ¨l,
2006). Several kinases that control NF-kB activation have been
identified(Ha ¨ckerandKarin,2006).Bycontrast, onlyafewphos-
phatases have been shown to regulate and terminate NF-kB
activity in a cell type-, pathway-, or substrate-specific fashion
(Chew et al., 2009; Eitelhuber et al., 2011; Li et al., 2006, 2008).
PP4R1 (protein phosphatase 4, regulatory subunit 1) was the
first noncatalytic, regulatory phosphatase subunit identified as
a constitutive interaction partner of the catalytic Ser-Thr
Immunity 37, 697–708, October 19, 2012 ª2012 Elsevier Inc. 697
phosphatase subunit PP4c (Kloeker and Wadzinski, 1999).
65% amino acid identity with PP2A catalytic subunits and
belongs to the PP2A-type phosphatases (Brewis et al., 1993).
including PP4R1, PP4R2, PP4R3a, PP4R3b, and PP4R4, which
give rise to a diverse collection of distinct PP4 holoenzymes
(Chen et al., 2008; Chowdhury et al., 2008; Gingras et al.,
2005; Lee et al., 2010).
Given the crucial role of NF-kB in lymphocyte physiology, it is
a number of lymphoid malignancies (Ruland and Mak, 2003b;
Skinnider and Mak, 2002). One very prominent example is cuta-
non-Hodgkin’s lymphomas (NHLs), which show addiction to
constitutively activated NF-kB (Hwang et al., 2008). The majority
of CTCL are represented by the entities of Mycosis fungiodes
(MF; tumorous skin manifestation) and the more aggressive Se ´z-
arise secondarily from late-stage MF or as de novo SS (Herling
et al., 2004; Hwang et al., 2008). MF and SS are slowly progres-
are difficult to treat and usually incurable. Although several
components of the NF-kB signaling machinery have been previ-
ously identified as bona fide oncogenes or tumor suppressors in
lymphocytes (Compagno et al., 2009; Lenz et al., 2008; Staudt,
2010), the molecular lesions driving aberrant NF-kB signaling in
CTCL remain largely enigmatic (Kiessling et al., 2011; Kim
et al., 2000).
Here, we report the identification of PP4R1 by a small inter-
fering RNA (siRNA) screen as a negative regulator of TCR and
TNFR1-dependent NF-kB activity in T lymphocytes. PP4R1
expression was triggered by T cell activation and proliferation
but lost in CTCL. PP4R1 bound to the inhibitor of NF-kB kinases
(IKK)a and IKKb and the catalytic subunit PP4c and thereby
directed PP4c phosphatase activity to the IKK complex. Conse-
quently, PP4R1 silencing caused sustained and increased IKK
activity and T cell hyperactivation. Furthermore, deficiency for
PP4R1 in CTCL resulted in constitutive IKK-NF-kB signaling
and thus formed an important molecular event maintaining the
malignant phenotype of a subset of CTCL cells. Our findings
show that PP4R1 is a regulator of IKK activity and a suppressor
RNAi Screen Identifies PP4R1 as a Regulator of NF-kB
To systematically uncover phosphatases that are involved in the
regulation ofNF-kBsignalinginTlymphocytes, weperformedan
RNAi-based screen using a subgenomic siRNA library. There-
fore, we engineered a Jurkat reporter T cell line that secretes
Gaussia luciferase in an NF-kB-dependent fashion (G-Luc
Jurkat; see Figures S1A–S1F available online). Primary RNAi
screening identifiedtwo PP4R1-targeting
augmented NF-kB reporter activity in response to TCR and to
TNFR1 stimulation (Figure 1A; Tables S1 and S2). In Jurkat
T cells, various independent siRNAs against PP4R1 led to
a detectable knockdown of PP4R1 and enhanced PMA+
ionomycin-induced upregulation and secretion of several
NF-kB-regulated cytokines (Figure 1B; Figures S1G–S1L).
Knockdown of PP4R1 elevated TCR+CD28-induced (Figure 1C)
or PMA- or TNFR1-induced (Figure 1D) NF-kB reporter activity
similarly to knockdown of the known negative regulator CYLD.
Furthermore, PP4R1 silencing enhanced expression of the
NF-kB target genes A20 and IkBa (data not shown) and of IL-2
and IL-8 (Figure 1E). The fact that PP4R1 impinges upon both
TCR- and TNFR1-induced NF-kB signaling indicated that
PP4R1 acts on a common level in canonical NF-kB signaling in
PP4R1 Expression Is Triggered by Lymphocyte
Activation and Expansion In Vitro
To evaluate the physiological relevance of our findings, wemoni-
tored PP4R1 expression in primary human peripheral blood T
lymphocytes. Surprisingly, PP4R1 was not detectable in resting
T cells. However, PP4R1 expression increased upon T cell acti-
vation and in vitro proliferation, whereas the expression of the
PP4 catalytic subunit PP4c remained constant (Figure 2A). The
activation-associated pattern of PP4R1 expression in human T
lymphocytes was highly reproducible and equally observed for
T cells that had been prestimulated in vitro either by the poly-
clonal mitogen PHA or by CD3+CD28 crosslinking using
agonistic antibodies (Figure 2B).
PP4R1 expression generally followed a pattern of lymphocyte
activation and differentiation. In fact, we observed a strong
PP4R1 positivity for activated centroblastoid B cells (Figures
S2A and S2B) and interfollicular T cells (Figures S2C and S2D)
in lymph node biopsies, whereas intrafollicular and perifollicular
T cells lacked PP4R1 expression (Figures S2F–S2L). As
observed in Jurkat T cells, PP4R1 knockdown in prestimulated,
expanded human T lymphocytes increased secretion of the
NF-kB-dependent cytokines IL-2, TNF-a, and IFN-g (Figure 2C).
Accordingly, knockdown of PP4R1 in prestimulated, expanded
human T lymphocytes drastically increased both basal and stim-
ulation-dependent NF-kB activity (Figure 2D), whereas ectopic
PP4R1 almost completely blunted NF-kB activation in T cells
PP4R1 Specifically Suppresses NF-kB Activation
stably expressing small hairpin RNAs (shRNAs) targeting PP4R1
(Figure S3A) and tested these cells for NF-kB, AP-1, and NF-AT
signaling by transient transfection of reporter genes. As ex-
pected, NF-kB activation was significantly elevated in PP4R1-
silenced cells, whereas AP-1 and NF-AT activation remained
unaffected (Figure 3A; Figure S3B). Importantly, enhanced
NF-kB activation of stably PP4R1-silenced cells was lost when
PP4R1 expression was transiently reconstituted (Figure 3B).
Moreover, exogenous PP4R1 decreased both basal and TCR+
CD28-induced NF-kB activity (Figures 3B and 3C, left), whereas
PP4R1 (Figure 3C). In addition, suppression of TCR+CD28-
dependent NF-kB activation by ectopically expressed PP4R1
occurred in a dose-dependent manner (Figure S3C). In line
with these findings, stably PP4R1-silenced Jurkat T cells dis-
played enhanced PMA+ionomycin-triggered expression of
multiple NF-kB target genes (Figure 3D) and increased secretion
of IL-2, IL-8, and IFN-g upon stimulation (Figure 3E; Figure S3D).
IKK Suppression by PP4R1-Driven Dephosphorylation
698 Immunity 37, 697–708, October 19, 2012 ª2012 Elsevier Inc.
instructions. For each 96-well transfection plate, a panel of positive and
negative control siRNAs encompassing oligos specific for CYLD, RelA,
Carma1, and TNF-R1 as well as nontargeting siRNA and mock transfections
(H2O) were included. Seventy-two hr posttransfection cells were either left
untreated or were stimulated using anti-CD3 (OKT3)/anti-CD28/goat anti-
mouse antibodies (0.1 mg/ml each) or recombinant TNF-a (20 ng/ml). Five hr
poststimulation Gaussia activity was measured in the cell culture supernatant
using a 96-well injection luminometer (Berthold Detection Systems) and
normalized to cell viability using the Celltiter-Glo assay (Promega). All
measurements were performed in duplicate.
Retroviral and Lentiviral Transductions
Retroviral supernatants were prepared by transfection of pMX IRES-GFP
expression vectors into amphotropic Phoenix cells (Swift et al., 2001). For
production of lentiviral particles, HEK293T cells, pretreated with 25 mM chlor-
oquin for 1 hr, were transfected with TRC-derived shRNA vectors and a
plasmid mixture for gag, pol, env and VSV-G for pseudotyping. Eight hr post
transfection medium was replaced from packaging cells. After 2 days, super-
natant was passed through a 0.45 mM filter, supplemented with Polybrene
(8 mg/ml). Target cells (1 3 105cells) were infected by spin occulation with
1.0 ml of viral supernatant. Stable integrants were selected by puromycin
resistance (1 mg/ml puromycin).
Retroviral Reconstitution Assays
The toxicity of ectopic phosphatase expression was examined by retroviral
reconstitution of the CTCL lines SeAx, MyLa, Hut78, and HH. For the reconsti-
tution assay, CTCL cell lines were retrovirally transduced with pMX-IRES GFP
vectors either expressing GFP alone (ctrl) or coexpressing WT human PP4R1,
PP4c, or PP2Ac. The frequency of GFP-positive cells was monitored over time
by flow cytometry. For a single assay, cells were transduced and analyzed in
triplicate. The percentage of phosphatase-reconstituted cells was normalized
to that of control-transduced cells at the same day. In addition, the ratio of
GFP-positive cells at various time points was normalized to the initial ratio at
day 3–5 posttransduction.
Reverse Transcriptase PCR and Quantitative Real-Time PCR
Total cellular RNA was isolated using the RNAqueous?-Micro Kit (Applied
Biosystems). Total RNA (0.5–1.0 mg) was reverse-transcribed with a reverse
formed with Power SYBR Green PCR Master Mix (Applied Biosystems). Gene
expression was analyzed using the 7.500 Real-Time PCR Systems and
Sequence Detection Software version 2.0.1 (Applied Biosystems). For some
experiments, Univeral Probe Libray (UPL) assays were designed and quantita-
tive RT-PCR was performed using the ProbesMaster Kit and the LightCycler?
1 (HPRT1) and/or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as
reference genes. Sequences of qPCR primers are available upon request.
Cell Culture, Transfections, and Reporter Assays
(Peter et al., 1997). For transient reporter assays, 1 3 107Jurkat T cells or 2 3
105293T cells were cotransfected with p33AP-1-Luc and pfos-LacZ or
pGL83NF-kB-fos and pfos-LacZ as described previously (Arnold et al.,
2001). Transfection efficiency was normalized to LacZ expression. Values
48 hr, cells were lysed in lysis buffer (30 mM Tris/HCl, pH 7.5, 150 mM NaCl,
2 mM EDTA, 1 mM PMSF, protease inhibitor cocktail [Roche], 1% Triton
X-100 (Serva), and 10% glycerol). For transient reporter assays, 1 3 107Jurkat
T cells were transfected by electroporation (250V, 950 mF) in 400 ml of IMDM
media using 10–30 mg plasmid DNA. For siRNA and cDNA transfections,
primary human T cells or Jurkat T cells were transfected by nucleofection
Immunoprecipitation, Immunoblotting, and In Vitro Kinase Assays
Cells were lysed using 1% NP-40 in 20 mM Tris pH 7.4 and 150 mM NaCl
supplemented with protease and phosphatase inhibitors. Two micrograms
of antibody coupled to Protein A sepharose was used to immunoprecipitate
proteins from cell lysates for 2–18 hr at 4?C. Proteins were resolved by SDS-
PAGE, transferred to Hybond nitrocellulose membrane (Amersham Pharmacia
Biotech) and processed according to the manufacturer’s protocol. In vitro
kinase assays were performed as described previously (Brenner et al.,
2005). Horseradish peroxidase (HRPO)-conjugated antibodies (Abs) were
purchased from Southern Biotechnology Associates. The Abs used were
anti-PP4R1, and anti-PP4c (all from Bethyl), anti-PP2Ac (Thermo Scientific),
anti-FLAG (M2, Sigma), anti-HA (3F10, Roche), anti-Erk, anti-b-actin (Sigma),
anti-IKKg (FL-419), and anti-PP1c (4G3) (all from Santa Cruz), anti-phospho-
Erk (BD Biosciences), anti-Carma1 (ProSci), anti-IKKb (Imgenex), anti-IKKa,
and anti-phospho-IKKa/b (Cell Signaling).
CTCL was diagnosed according to the WHO-EORTC classification of cuta-
neous lymphomas and the criteria of the International Society of Cutaneous
Lymphomas. This study includes patients with MF and SS seen at Mannheim
University Medical Centre and the University Hospital Cologne and was
performed in accordance with the ethical guidelines of the German Cancer
Research Center (DKFZ, Heidelberg) and with the provisions of the Helsinki
protocol. Patients signed the institutional consent forms for use of tissue for
research. In total, 32 CTCL patients were included for analysis of PP4R1
expression by qPCR (n = 12), immunoblot analysis (n = 12), or IHC analysis
of skin biopsies (n = 8).
Mean and SD for all quantitative measurements are representative of triplicate
measurements. In order to examine statistical significance the Student’s t test
(independent two-sample t test, unequal variance) was used. A p value < 0.05
was considered as statistically significant. Overall, the level of statistical
significance was defined as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
Supplemental Information includes seven figures, four tables, and Supple-
mental Experimental Procedures and can be found with this article online at
We thank N. Oberle and E.-M. Weiß for technical advice, D. Brenner for critical
discussion of the data, and H. Sauter for excellent secretarial work. This work
was supported in part by the Cooperation Program in Cancer Research of
the Deutsches Krebsforschungszentrum (DKFZ) and the Israeli Ministry of
Science, Culture and Sport (MOST), the Deutsche Krebshilfe, the Wilhelm
Sander Stiftung, the SFB 405, and the Tumorzentrum Heidelberg/Mannheim.
M.Boutros acknowledges support of the Helmholtz Alliance for Systems
Biology and by an Emmy-Noether Grant of the Deutsche Forschungsgemein-
schaft. R.A., M.Brechmann, T.M., M.H., and N.W. are supported by the Jose ´
Carreras Leuka ¨mie-Stiftung, J.P.N. received funding from the Helmholtz
Alliance for Immunotherapy of Cancer, and M.H. receives funding through
the Max-Eder Junior Group Program of the German Cancer Aid.
Received: June 7, 2011
Accepted: July 9, 2012
Published online: October 18, 2012
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