Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α ...

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Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase * Guisheng Zhou§¶, Kathie A. Mihindukulasuriya§¶, Rebecca A. MacCorkle-Chosnek§, Aaron Van Hooser!, Mickey C.-T. Hu#, B. R. Brinkley!, and Tse-Hua Tan§² From §the Department of Immunology and ‡the Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030; #the Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030. Running Title: Positive Regulation of the JNK Pathway by PP4 * This work was supported by NIH grants AI-38649 and AI-42532 (to T.-H. Tan) and CA-41424 (to B. R. Brinkley). K. A. Mihindukulasuriya and R. A. MacCorkle-Chosnek are recipients of United States Army Breast Cancer Research Program Predoctoral Fellowships DAMD 17-011-0139 and DAMD 17- 00-1-0141, respectively. 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. ¶ These authors contributed equally to this work. ²A Scholar of the Leukemia & Lymphoma Society. To whom correspondence should be addressed: Department of Immunology, Baylor College of Medicine, M929, One Baylor Plaza, Houston, Texas 77030. Tel: 713-798-4665; Fax: 713-798-3033; E-mail: Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on November 6, 2001 as Manuscript M107014200 by guest on April 12, 2018 http://www.jbc.org/ Downloaded from

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Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced

Activation of c-Jun N-terminal Kinase*

Guisheng Zhou§¶, Kathie A. Mihindukulasuriya§¶, Rebecca A. MacCorkle-Chosnek§, Aaron

Van Hooser!, Mickey C.-T. Hu#, B. R. Brinkley!, and Tse-Hua Tan§†

From §the Department of Immunology and ‡the Department of Molecular and Cellular Biology, Baylor

College of Medicine, Houston, Texas 77030; #the Department of Molecular and Cellular Oncology, The

University of Texas M.D. Anderson Cancer Center, Houston, TX 77030.

Running Title: Positive Regulation of the JNK Pathway by PP4

*This work was supported by NIH grants AI-38649 and AI-42532 (to T.-H. Tan) and CA-41424 (to

B. R. Brinkley). K. A. Mihindukulasuriya and R. A. MacCorkle-Chosnek are recipients of United States

Army Breast Cancer Research Program Predoctoral Fellowships DAMD 17-011-0139 and DAMD 17-

00-1-0141, respectively. 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.

¶ These authors contributed equally to this work.

†A Scholar of the Leukemia & Lymphoma Society. To whom correspondence should

be addressed: Department of Immunology, Baylor College of Medicine, M929, One

Baylor Plaza, Houston, Texas 77030. Tel: 713-798-4665; Fax: 713-798-3033; E-mail:

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 6, 2001 as Manuscript M107014200 by guest on A

pril 12, 2018http://w

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[email protected].

1The abbreviations used are: TNF-α, tumor necrosis factor-α; PP4, protein phosphatase 4; MAPK,

mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated

protein kinase; ERK, extracellular signal-regulated kinase; SDS-PAGE, SDS-polyacrylamide

gel electrophoresis; HA, hemagglutinin; HEK293, human embryonic kidney 293.

ABSTRACT

Protein phosphatase 4 (PP4, previously named protein phosphatase X [PPX]), a highly conserved, PP2A-related,

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novel serine/threonine phosphatase, has been shown to be involved in essential cellular processes, such as

microtubule growth and NF-κB activation. We provide evidence here that PP4 is involved in TNF-α

signaling in human embryonic kidney 293T (HEK293T) cells. Treatment of HEK293T cells with

TNF-α resulted in time-dependent activation of endogenous PP4, peaking at 10 min, as well as

increased serine and threonine phosphorylation of PP4. We also found that PP4 is involved in

relaying the TNF-α signal to JNK as indicated by the ability of PP4-RL, a dominant-negative

PP4 mutant, to block TNF-α-induced JNK activation. Moreover, the response of JNK to TNF-

α was inhibited in HEK293 cells stably expressing PP4-RL in comparison to parental HEK293

cells. The involvement of PP4 in JNK signaling was further demonstrated by the specific

activation of JNK, but not p38 and ERK2, by PP4 in transient transfection assays. However, no

direct PP4-JNK interaction was detected, suggesting that PP4 exerts its positive regulatory effect

on JNK in an indirect manner. Taken together, these data indicate that PP4 is a signaling

component of the JNK cascade and involved in relaying the TNF-α signal to the JNK pathway.

INTRODUCTION

A major mechanism by which cells regulate protein function is to add or remove phosphate groups on serine,

threonine and tyrosine residues. The steady-state level of phosphorylation, and thus the strength and duration of the

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signal transmitted, are balanced by the opposing actions of protein kinases and protein phosphatases (1-3). Protein

kinases, protein phosphatases, and their substrates are integrated within an elaborate signal transducing network (3-

5). The defective or inappropriate operation of this network leads to many diseases such as cancer, diabetes, and

autoimmune disorders (6).

Mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated kinase (ERK), c-Jun N-

terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, play essential roles in many important

biological processes such as the stress response, cell proliferation, apoptosis, and tumorigenesis (7-9). MAPK

activation involves sequential protein kinase reactions within a three-kinase module (MAP3K-MAP2K-MAPK),

whereby a MAP3K phosphorylates and activates a MAP2K, a dual-specificity kinase, that then phosphorylates and

activates a MAPK (7,8,10). In vivo MAPK phosphorylation is a reversible process, indicating that protein

phosphatases provide an additional level of regulation of MAPKs. In fact, the magnitude and duration of MAPK

activation are tightly controlled by the coordinate actions of protein kinases and protein phosphatases. A large

number of mammalian MAPK phosphatases (MKPs) have been identified, including dual-specificity phosphatases

and tyrosine-specific phosphatases (11,12). There is evidence that serine/threonine-specific phosphatases also

regulate MAPKs (13,14). MKPs inactivate MAP kinases by directly dephosphorylating both threonine and tyrosine

residues of MAPKs (12). The coordinate regulation by protein kinases and phosphatases also occurs at many other

points within the three-kinase module. For example, MKP-1, a dual-specificity phosphatase, inhibits ERK, but

positively regulates Raf-1 and MKK in an ERK-independent manner (15). PP2A also acts on multiple components

of the ERK pathway (12).

Protein phosphatase 4 (PP4, previously named protein phosphatase X [PPX]) is a novel protein serine/threonine

phosphatase that is a member of the PP2A family of phosphatases (16). PP4 is highly conserved during evolution,

with human and Drosophila PP4 sharing 91% amino acid identity (16). It has been shown that PP4 is localized at the

centrosomes in mammalian cells and Drosophila embryos, and that PP4 is involved in the regulation of microtubule

growth/organization at centrosomes (17,18). Our previous studies showed that PP4 interacts with members of the

NF-κB family, such as c-Rel, p50, and RelA, stimulates the DNA-binding activity of c-Rel, and

activates NF-κB-mediated transcription (19). The high degree of conservation of PP4 suggests

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that PP4 may be involved in many more essential cellular processes and is tightly controlled in

vivo. It has been shown that PP4 is carboxymethylated (20). Furthermore, three potential

regulatory subunits have been identified for PP4: α4 (21,22), PP4R1 (23), and PP4R2 (18). In an

effort to further investigate the cellular function of PP4, we found that PP4 acts as a specific

positive regulator for the JNK pathway and that PP4 is required to relay the TNF-α signal to the

JNK pathway.

MATERIALS AND METHODS

Reagents[γ-32P]ATP and [32P]orthophosphate were purchased from ICN Biomedicals

(Irvine, CA). An enhanced chemiluminescence system was purchased from Amersham

Pharmacia Biotech (Piscataway, NJ). Ser/Thr phosphatase assay kit 1 was purchased from

Upstate Biotechnology, Inc. (Waltham, MA). TNF-α was purchased from R&D Systems

(Minneapolis, MN). Anti-HA antibody (12CA5) was purchased from Boehringer-Mannheim

(Indianapolis, IN). Monoclonal anti-Flag (M2) and anti-γ-tubulin antibodies were purchased

from Sigma (St. Louis, MO). Monoclonal anti-PP1 and anti-c-Myc (9E10) antibodies, and goat

anti-Bcl-XL antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat

anti-aldolase antibody was purchased from Biodesign (Saco, ME). Monoclonal anti-Golgin-97

was purchased from Molecular Probes (Eugene, OR). Rabbit anti-GRP78 polyclonal antibody

was purchased from StressGen (Victoria, BC Canada). Monoclonal anti-Lamin B1 was

purchased from ZYMED Laboratories (So. San Francisco, CA). Goat anti-human and -rabbit

IgG (H+L) conjugated to FITC and Texas Red were purchased from Jackson Immunoresearch

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Laboratories, Inc. (West Grove, PA). Rabbit anti-PP4 polyclonal antibodies Ab104 and Ab6101

were raised against the C-terminal regions of PP4, EAAPQETRGIPSKKPVADY287-305, and

QETRGIPSKKPVA291-303, respectively. Ab104 and Ab6101 were peptide purified using the

Sulfolink Kit from Pierce (Rockford, IL). Rabbit anti-JNK1 polyclonal antibody (Ab101) was

described previously (24). Human autoimmune serum (#4171, 1:2000) specific for proteins of

the pericentriolar matrix was described previously (25). All other chemical reagents were

purchased from Sigma (St. Louis, MO) unless otherwise noted.

PlasmidsThe GST-Jun (1-79) was a gift from Dr. M. Karin (UCSD, CA). GST-ATF2 (1-

96) and pHA-ERK2 were provided by Dr. J. S. Gutkind (National Institutes of Health, Bethesda,

MD). GST-JNK (also called GST-SAPK) was a gift from Dr. L. I. Zon (Children’s Hospital,

Boston, MA). pHA-MKK6 was provided by Dr. Z. Yao (Amgen, Boulder, CO). pHA-PKC-ζ,

was a gift from Dr. M. W. Wooten (Auburn University, AL). pHA-JNK1 and HA-p38 were

gifts from Dr. J. Woodgett (Ontario Cancer Institute, Toronto, Canada). pCMV-PP1 was a gift

from Dr. A. H. Schonthal (University of Southern California, Los Angeles, CA) (26). pMTSM-

Myc-M3/6 was a gift from Dr. K. E. Davis (University of Oxford, Oxford, UK) (27). pBJF-

Flag-PP2A and pBJF-Flag-PP6 were kindly provided by Dr. J. Chen (University of Illinois at

Urbana-Champaign, IL) (21). pCIneo-Flag-PP4 was constructed by inserting an XbaI site and a

Flag tag at the 5’-end and a NotI site at the 3’-end of full-length human PP4 cDNA (19) and

subcloning the PCR product into the pCIneo expression vector. PP4 and HA-PP4-RL were

described previously (19).

Cells and transfection Human HeLa cells, human embryonic kidney 293T (HEK293T) and

293 (HEK293) cells were obtained from the American Type Culture Collection (Rockville, MD)

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and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf

serum and 100 U/ml streptomycin/penicillin at 37oC in a humidified atmosphere of 5% CO2.

HEK293T cells were plated at a density of either 1.5 x 105 cells per 35-mm plate well or 1.5 x

106 cells per 100-mm dish and transfected the next day using the modified calcium phosphate

precipitation protocol (Specialty Media, Inc., Lavallette, NJ). Cells were transfected with

plasmids encoding β-galactosidase (0.15 µg) in combination with an empty vector or various

amounts of plasmids encoding phosphatases, phosphatase mutants, kinases, or kinase mutants as

indicated in the figure legends.

Coimmunoprecipitation, immunocomplex kinase assays, and Western blot

analysisCoimmunoprecipitation and immunocomplex kinase assays were performed as described previous

(28-31). Western blot analysis was performed using an enhanced chemiluminescence detection

kit according to the manufacturer’s protocols (Amersham).

Phosphatase assaysHEK293T cells were lysed in buffer containing 50 mM Tris-HCl

[pH 8.0], 1% Nonidet P-40, 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 1 mM

dithiothreitol, 50 µM p-amidinophenyl methanesulfonyl fluoride (PMSF), and 2 µg/ml

aprotinin. Endogenous PP4 was immunoprecipitated with an anti-PP4 (Ab104) antibody.

Overexpressed Flag-PP4 and HA-PP4-RL were immunoprecipitated with anti-Flag

(M2) and anti-HA (12CA5) antibodies, respectively. The immunoprecipitates were

washed three times with buffer containing 50 mM HEPES [pH 7.4], 0.1% Triton X-100,

and 500 mM NaCl. Phosphatase assays were performed using Ser/Thr phosphatase assay

kit 1, according to the manufacturer’s protocol (Upstate Biotechnology, Inc., Waltham,

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MA). The immunoprecipitates were incubated with 4 µM KTpIRR peptide in 40 µl of

assay buffer (50 mM Tris [pH 7.0], 0.1 mM CaCl2, and 1 mM MnCl2) at 30ºC for 30

min (unless otherwise indicated in the figure legend). Buffer plus peptide was used as a

negative control. The immunoprecipitates were then pelleted and the assay buffer was

transferred to a 96-well, half-volume plate. The assay was terminated by the addition of

100 µl of Malachite Green solution (1 volume of 4.2% (w/v) ammonium molybdate in 4

M HCl, 3 volumes of 0.045% (w/v) Malachite Green in water, and 1 µl per ml of 10%

Tween-20 added fresh). After 15 min at room temperature, the assay was read at 650 nm

on a Perkin Elmer Bioassay Reader (HTS 7000 plus).

In vitro binding assaysGST and GST-SAPK fusion protein were immobilized on

glutathione-Sepharose 4B beads equilibrated in incubation buffer containing 20 mM Tris-HCl

[pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM

phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 2 µg/ml aprotinin. Cell lysates (600 µg)

from HEK293 cells stably transfected with Flag-PP4 or HEK293T cells transiently transfected

with Myc-M3/6 were incubated with GST-JNK fusion protein or GST-4T-Sepharose beads in

incubation buffer containing 3 mg/ml bovine serum albumin at 40C for 2 h. The beads were

washed five times with the incubation buffer, boiled in a SDS-PAGE loading buffer for 5 min,

resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and then subjected to

Western blotting with an anti-Flag (M2) or an anti-Myc antibody. The membrane was then

stripped with stripping buffer (62.5 mM Tris-HCL [pH 6.7], 100 mM 2-mercaptoethanol, 2%

SDS) and reprobed with an anti-GST antibody.

Centrosome isolationCentrosomes were purified from HeLa cells by a standard protocol

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(32,33). Briefly, 6 x 107 HeLa cells were incubated with 0.2 µM Nocadazole and 1 µg/ml

Cytochalasin D at 37°C for 60 min. After trypsinization, the cells were pelleted and washed one

time with 1X TBS (50 mM Tris [pH 7.6], 150 mM NaCl) and one time with 0.1X TBS + 8%

sucrose. The cells were then resuspended in 2 ml of 0.1X TBS + 8% sucrose and lysed by adding

8 ml of fractionation lysis buffer (1 mM HEPES [pH 7.2], 0.5% NP-40, 0.5 mM MgCl2, 0.1%

β-mercaptoethanol, 1 µg/ ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF, 1 mM Na3VO4, and 0.5

mM NaF). The lysate was spun at 2,500 g for 10 min. The supernatant was collected and spun

again at 2,500 g for 10 min. The supernatant was transferred into a new tube through a 70 µm

nylon filter (Falcon #2350). The resulting supernatant was incubated with 10 mM HEPES and 1

µg/ml DNase on ice for 30 min, transferred to a 15 ml ultracentrifuge tube, underlaid with 1 ml of

60% sucrose in sucrose dilution buffer (10 mM PIPES [pH 7.2], 0.1% Triton X-100, and 0.1%

β-mercaptoethanol), and spun at 10,000 g for 1.5 h. The bottom 3 ml was transferred to a new tube

containing a discontinuous 40/50/70% sucrose gradient in sucrose dilution buffer. After spinning

at 120,000 g for 1.5 h, 0.5 ml fractions from the top were taken and diluted to 1 ml with 0.5 ml

PEM buffer (80 mM PIPES [pH 6.8], 5 mM EGTA, 2 mM MgCl2). After mixing, the solution

was spun at 15,000 rpm in a tabletop centrifuge for 30 min. The pellet was resuspended in 1 ml

of PEM buffer and spun at 15,000 rpm in a tabletop centrifuge for 30 min. The final pellet

containing centrosomes was washed twice with PEM buffer and then resuspended in Laemelli

sample buffer (Bio-Rad, Hercules, CA) with 5% β-mercaptoethanol.

ImmunofluorescenceAs recently described in detail (34), cells were grown on

coverslips and the coverslips were washed in 0.5% Triton X-100 for 2 min and fixed in

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cold 4% ultrapure formaldehyde (Polysciences, Inc.) in PEM buffer (80 mM K-PIPES

[pH 7.0], 5 mM EGTA, 2 mM MgCl2) for 10-20 min. For the immunofluorescence of

β-tubulin, 4% polyethylene glycol was added to PEM buffer during the permeabilization

and fixation steps. After they were fixed, the coverslips were washed with PEM buffer

and permeabilized in 0.5% Triton X-100 in PEM buffer for 30 min. Then, the coverslips

were washed with PEM buffer and blocked in 2.5% non-fat dry milk in TBST buffer (50

mM Tris [pH 7.6], 150 mM NaCl, 0.1% Tween-20) overnight. The next day, the

coverslips were incubated for 1 h at 37oC with primary antibodies diluted in TBST,

washed in TBST, and incubated for 1 h at 37oC with secondary antibodies diluted in

1:200 in TBST. After washing in TBST, coverslips were counterstained with 0.4 µg/ml

4,6 diamino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR) in TBST and

mounted with VectashieldR antifade medium (Vector Laboratories, Burlingame, CA) or

ProLong antifade medium (Molecular Probes, Eugene, OR). The figures are composite

images obtained with a Deltavision, deconvolution-based optical workstation (Applied

Precision, Issaquah, WA). Z-series stacks of multiple focal planes were used to render

3-D volumes.

Establishment of HEK293 cell clones stably transfected with Flag-PP4 and HA-PP4-

RLHEK293 cells were grown in a complete DMEM medium containing 10% fetal calf serum

supplemented with 12.5 mM HEPES, 50 µg/ml gentamycin and 100 U/ml penicillin-

streptomycin (GIBCO-BRL). We transfected the HEK293 cells with Flag-PP4 by the Fugene 6

method according to the manufacturers protocol (Roche Molecular Biochemicals, Indianapolis,

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IN). The transfected cells were selected by Geneticin (G418; GIBCO-BRL, Rockville, MD) at a

concentration of 750 µg/ml or 1 mg/ml. The cells were replated at a 1:15 dilution whenever they

reached 80% confluence. After 10 to 14 days, the T-75 flasks were trypsinized, and the drug-

resistant cells were replated at a limiting dilution to obtain independent clones. Each clone was

tested for Flag-PP4 expression by Western blotting. A similar approach was used to establish an

HEK293 cell line stably expressing HA-PP4-RL.

In vivo labeling of PP4 and phosphoamino acid analysisHEK293T cells (1 x 106 cells in

100 mm dishes) were transfected with 5 µg of Flag-PP4. After 40 h, the cells were maintained in

phosphate-free DMEM medium containing 5% dialyzed serum for 1 h at 37oC. The cells were

then labeled in phosphate-free DMEM medium supplemented with 5% dialyzed serum and 100

µCi [32P]orthophosphate/ml for 4 h at 37oC. After TNF-α treatment, the cells were washed with

PBS twice to remove free [32P]orthophosphate. Flag-PP4 was immunoprecipitated with an anti-

Flag antibody (M2) and separated by SDS-PAGE. The separated proteins were transferred to

PVDF, and autoradiography was performed. The membrane was then subjected to

immunoblotting using an anti-Flag (M2) antibody. The corresponding PP4 bands were cut out

and subjected to phosphoamino acid analysis (35,36).

RESULTS

PP4 is activated by TNF-α In an effort to investigate which signaling pathway(s)

PP4 may be involved in, we examined the effect of TNF-α on PP4 phosphatase activity.

We first generated an anti-PP4 antibody, Ab104, which recognizes the C-terminal

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region of PP4. Western blot analysis indicated that Ab104 specifically recognized PP4,

but not the most highly homologous phosphatases PP2A and PP6 (Fig. 1A). Previously,

PP4 had been shown to localize to the centrosomes via immunofluorescence staining

(17,18). To confirm the specificity of Ab104, we isolated centrosomes from HeLa cells

and performed Western blotting with antibodies to PP4 (Ab104), as well as markers for

various subcellular compartments. PP4 localized to centrosome fractions, and these

fractions were shown to be free of contamination from other subcellular compartments

(Fig. 1B). We noticed that PP4 did not peak with γ-tubulin. Considering that γ-tubulin is

a component of the centrioles of the centrosomes and that PP4 has been previously

reported to be a component of the pericentriolar matrix of the centrosomes (17), the slight

difference in the Western blot detection may be the result of slight differences in the

densities of the two centrosomal structures. The association of PP4 with the centrosome

was further confirmed by immunofluorescence staining using a peptide purified anti-PP4

antibody (Ab104). As shown in Fig. 1C, PP4 co-localized with proteins of the

pericentriolar matrix (PCM). Taken together, these data show that PP4 is a component of

the centrosome.

We then measured the phosphatase activity of PP4 before and after TNF-α treatment.

PP4 phosphatase assays were established by using a synthetic peptide substrate, KTpIRR.

We first wanted to ensure that the PP4 phosphatase assay is able to measure PP4

phosphatase activity. So, we tested the assay to determine the linear range of the assay

and to show that increasing amounts of PP4 correlate with increasing PP4 activity. PP4

showed a time-dependent increase in its phosphatase activity in a time period of 1 to 50

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min of incubation of PP4 with the peptide substrate (Fig. 2A, upper panel). Within this

time frame, PP4 activity increased with increased amounts of PP4 (Fig. 2A, lower panel).

HEK293T cells were treated with TNF-α (10 ng/ml), and endogenous PP4 was

immunoprecipitated from the cells with the PP4-specific antibody, Ab104. The PP4

phosphatase activity was measured by incubating the immunoprecipitated PP4 with the

peptide substrate, KTpIRR, for 30 min. PP4 phosphatase activity was increased following

TNF-α stimulation in a time-dependent fashion, peaking at 10 min (Fig. 2B, upper

panel). PP4 activity was decreased after 10 min, indicating that TNF-α-induced PP4

activation was a transient event. The increased phosphatase activity was not due to

variation in levels of PP4 since the amounts of PP4 immunoprecipitated were comparable

(Fig. 2B, lower panel). Therefore, PP4 was activated in response to TNF-α in HEK293T

cells.

It is known that TNF-α is a potent activator of the JNK pathway. To establish a

possible link between PP4 and the JNK pathway in response to TNF-α, endogenous JNK

was immunoprecipitated with an anti-JNK1 antibody (Ab101) from HEK293T cells, and

its kinase activity was determined by an immunocomplex kinase assay using GST-cJun

(1-79) as substrate. As shown in Fig. 2C, JNK was activated by TNF-α with similar

kinetics to that of PP4 in HEK293T cells. Thus, PP4 was activated concomitant with JNK

activation in response to TNF-α in HEK293T cells.

TNF-α induces serine and threonine phosphorylation of PP4To further confirm the

involvement of PP4 in TNF-α signaling, we examined the effect of TNF-α on the

phosphorylation state of PP4, since PP2A, the phosphatase most homologous to PP4, is

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regulated by phosphorylation. HEK293T cells were transfected with Flag-PP4, labeled in

vivo with [32P]orthophosphate, and treated with TNF-α (10 ng/ml). Flag-PP4 was then

immunoprecipitated with an anti-Flag antibody (M2). We found that TNF-α treatment

induced phosphorylation of PP4 in a time-dependent manner, peaking at 5 min (Fig. 3A).

Phosphoamino acid analysis showed that TNF-α-induced phosphorylation of PP4

occurred on serine and threonine residues (Fig. 3B). These results indicate that PP4 is

inducibly phosphorylated in response to TNF-α in HEK293T cells.

JNK activation by TNF-α is blocked by PP4-RLTo investigate the functional

involvement of PP4 in the TNF-α signaling, we examined the contribution of PP4 to

TNF-α-induced JNK activation. We first constructed a PP4 mutant, PP4-RL, in which

the replacement of arginine-236 with leucine resulted in the loss of its phosphatase

activity (Fig. 4B). We then examined the effect of PP4-RL on JNK activation by TNF-

α. HEK293T cells were transfected with HA-JNK1 alone or HA-JNK1 plus PP4-RL. The

transfected cells were treated with TNF-α (10 ng/ml) for 10 min. We found that TNF-

α-induced JNK activation was blocked by PP4-RL (Fig. 4A, upper panel), indicating that

PP4-RL may be a dominant-negative mutant and that PP4 plays a role in JNK activation

by TNF-α.

We also established a HEK293 cell clone, called HEK293-PP4-RL, that stably

expresses HA-PP4-RL (Fig. 4C, right panel). HEK293-PP4-RL cells were treated with

TNF-α (10 ng/ml) for various times (0 to 60 min), and endogenous JNK was

immunoprecipitated from the cells with an anti-JNK antibody (Ab101). The JNK kinase

activity was measured by immunocomplex kinase assays using GST-cJun (1-79) as a

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substrate. As shown in Fig. 4C (left panel), a decrease in JNK activation by TNF-α in

HEK293-PP4-RL cells was detected, in comparison to the parental HEK293 cells.

While JNK activation by TNF-α peaked at 10 min in HEK293T cells (Fig. 2C), TNF-α-

induced JNK activation peaked at 20 min in HEK293 cells (Fig. 4C, left panel). This

kinetic difference between HEK293 and HEK293T cells may be due to the presence of

SV40 large T antigen in HEK293T cells. Taken together, these data indicate that PP4 is

required for transducing TNF-α signals to the JNK pathway.

PP4 specifically activates JNK, but not p38 and ERK2To confirm the involvement

of PP4 in the JNK signaling pathway, we tested whether expression of PP4 had any effect

on the activity of JNK. Hemagglutinin (HA)-tagged JNK1 was cotransfected in

HEK293T cells with PP4, PP1, another serine/threonine phosphatase, or M3/6, a dual-

specificity MAPK phosphatase. HA-JNK1 was immunoprecipitated, and its kinase

activity was determined in vitro using GST-cJun (1-79) as a substrate. Cotransfection of

PP4 resulted in activation of JNK1 (Fig. 5, lanes 1 and 2), whereas, PP1 and M3/6 had no

such effect on JNK1 (Fig. 5, lanes 1, 3, and 4). M3/6 is a known JNK-inactivating dual-

specificity phosphatase, which dephosphorylates the TPY motif of JNK (27,37).

Transiently transfected JNK is somehow partially activated. Therefore, cotransfection of

M3/6 with JNK resulted in inhibition of JNK activity, as expected. The nature of the

inhibition of JNK by PP1 is not known at this point. It is likely that PP1 dephosphorylates

the threonine residue of the TPY motif of JNK and thus inhibits JNK activity. These data

indicate that PP4 exerted a positive regulatory effect on JNK1.

To determine whether PP4’s effect on JNK1 is specific, we also examined the effect of

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PP4 on p38 and ERK2. HEK293T cells were transfected with various amounts of the PP4

expression plasmid together with the hemagglutinin (HA)-tagged MAPK constructs,

HA-JNK1, HA-p38, and HA-ERK2. HA-tagged MAPKs were immunoprecipitated,

and their kinase activities were determined in vitro using the appropriate substrates

(GST-cJun for JNK1, GST-ATF2 for p38, and MBP for ERK2). JNK was activated by

PP4 in a dose-dependent manner by PP4 (Fig. 6A). In contrast, PP4 had no significant

effect on the activities of either p38 (Fig. 6B) or ERK2 (Fig. 6C). These data indicate that

PP4 serves as a specific positive regulator for the JNK signaling pathway. We also found

that PP4-RL had no effect on PKC-ζ-induced ERK and MKK6-induced p38 activation

(data not shown).

We next wanted to determine whether PP4 and JNK1 interact directly with each other.

We incubated GST-JNK fusion protein with cell lysates from untreated or TNF-α

treated HEK293 cells stably expressing Flag-PP4. The potential PP4-JNK interaction

was analyzed by SDS-PAGE and Western blotting using an anti-Flag antibody (M2).

Similar to transient transfected Flag-PP4 in HEK293T cells (Fig. 3), stably expressed

Flag-PP4 in HEK293 cells was also inducibly phosphorylated after 5-min treatment of

TNF-α (Fig. 7A, lower panel). Association of PP4 with GST-JNK was not detectable

(Fig. 7A, upper panel) in the absence or presence of TNF-α. We also found that PP4 had

no phosphatase activity toward in vitro phosphorylated GST-JNK (data not shown).

Under the same conditions, however, M3/6, a dual-specificity phosphatase known to

target JNK directly, interacted with GST-JNK (Fig. 7B). Taken together, these data

suggest that PP4 affects the JNK pathway in an indirect manner.

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DISCUSSION

TNF-α is an important effector cytokine for inflammatory and immune responses and is

involved in many important cellular processes, such as proliferation, differentiation, and

apoptosis (38). A variety of protein phosphatases have been implicated in TNF-α signaling. For

example, calcineurin, a calcium-dependent serine/threonine phosphatase, participates in TNF-

α-mediated apoptosis in rat hepatoma cells (39) and SHP-2, a Src homology (SH)2-containing

phosphotyrosine phosphatase, mediates the induction of interleukin (IL)-6 by TNF-α through

modulation of the nuclear factor κB (NF-κB) pathway (40). Another phosphotyrosine

phosphatase, SHP-1, has been shown to mediate TNF-αs inhibitory effect on vascular

endothelial cell growth factor (VEGF)-induced endothelial cell proliferation (41). PP2A has also

been shown to be involved in many TNF-α-induced cellular processes (42-45). However, many

of these studies relied on the use of okadaic acid, an inhibitor for PP1 and PP2A. Since okadaic

acid inhibits PP4 with an IC50 comparable to that of PP2A (17), it is necessary to reexamine

some of the functions assigned to PP2A. We provide evidence here that PP4, a novel member of

the PP2A family, was activated by TNF-α in HEK293T cells, as indicated by increased

phosphatase activity, and increased serine and threonine phosphorylation of PP4 itself. The

involvement of PP4 in TNF-α signaling was further demonstrated by the observation that a PP4

mutant blocked TNF-α-induced JNK activation. Demonstration of the involvement of PP4 in

TNF-α signaling will help in exploring the molecular mechanism by which TNF-α regulates

cellular processes.

We found that the activation of PP4 by TNF-α was accompanied by an increase in the serine

and threonine phosphorylation of PP4. These results indicate the novel finding that a member of

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the PP2A family is subject to regulation by serine phosphorylation. It has been known that the

catalytic subunit of PP2A is subject to phosphorylation of a conserved tyrosine and an as yet

unidentified threonine (46-48), and that phosphorylation of either the tyrosine or the threonine

site inhibits phosphatase activity of PP2A in vitro. However, in human hepatoma Hep3B cells,

interleukin-6 (IL-6) induced an increase in both the phosphorylation and phosphatase activity of

PP2A (39). The nature of PP4 serine and threonine phosphorylation in response to TNF-α

remains unknown at this point. We noted that PP4 phosphorylation preceded PP4 activation in

response to TNF-α (5 min vs. 10 min). Considering the existence of multiple potential

phosphorylation sites on PP4, we speculate that PP4 may be subject to multiple phosphorylation

in response to TNF-α, and it is the phosphorylation that occurred at 10 min, but not at 5 min,

that contributes to activation of PP4. Further study, including identification of the

phosphorylation site(s) and characterization of site-directed mutants of PP4, is required to

understand the relationship between the phosphorylation, which occurred at 5 min, and PP4

activation. Alternatively, we cannot exclude the possibility that PP4 phosphorylation precedes

PP4 activation by inducing conformational change(s) and/or recruiting some regulatory subunits

required for the activation of PP4.

Phosphorylation-dependent inactivation is characteristic of many types of protein kinases,

such as DNA-dependent protein kinase (49), phosphinositide 3 kinase (50), Raf-1 (51-53), and

CLK1 (54). It has been shown that PP2A dephosphorylates the inhibitory phosphoserine residue

259 of Raf-1 and thus serves as a positive regulator for Raf-1, an upstream activating kinase for

the ERK pathway (55). Raf-1 and MEK1/2, another upstream activating kinase for the ERK

pathway, are positively regulated by MAP kinase phosphatase 1, a dual-specificity phosphatase,

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in an ERK-independent manner (15). We provide evidence here that PP4 acts as a specific

positive regulator for the JNK pathway. However, we did not detect a direct interaction between

PP4 and JNK1, strongly suggesting that PP4 exerts its positive regulatory effect on the JNK

pathway in an indirect manner. Given the fact that the core of the JNK signaling pathway is a

multiple-kinase module that is assembled by scaffold proteins to act as a stimulus-specific

signaling complex (7-9), and that the magnitude and duration of JNK activation are tightly

controlled by the coordinate actions of protein kinases and protein phosphatases (12), we

speculate that PP4 may target and activate the JNK upstream activating kinase(s), which is

negatively regulated by phosphorylation, and subsequently leads to JNK activation. The target

for PP4 could be a kinase at one or multiple levels of the JNK signaling cascade.

In addition to regulation of upstream activating kinases, we cannot exclude the possibility that

PP4 may target a phosphatase which inhibits JNK, and thus exert an indirect positive effect on

the JNK pathway. This putative JNK phosphatase may be activated by phosphorylation, and

hence inactivated by dephosphorylation. Since only JNK, but not p38 or ERK, is activated by

PP4, the putative phosphatase should also be JNK-specific. Inhibition of this JNK-specific

phosphatase by PP4-mediated dephosphorylation would then lead to JNK activation. Therefore,

some JNK-specific, dual-specificity phosphatases, such as M3/6 (37), may be good candidates

for PP4 targets.

AcknowledgementsWe would like to thank our colleagues for providing valuable reagents;

members of the Tan laboratory for helpful discussions and critical reading of the manuscript; Dr.

C. H. McDonald for assistance in phosphoamino acid analysis; A. Ashtari for technical

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assistance; and S. Robertson for secretarial assistance.

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FIGURE LEGENDS

FIG. 1. Characterization of a PP4-specific antibody, Ab104. A, the anti-PP4 antibody,

Ab104, specifically recognizes PP4, but not PP2A and PP6. HEK293T cells were

transfected with 2 µg of empty vector (lanes 1), 2 µg of Flag-PP4 (lanes 2), 2 µg of

Flag-PP2A (lanes 3), or 2 µg of Flag-PP6 (lanes 4). Cells were harvested 36 h after

transfection and subjected to SDS-PAGE. Western blotting was performed with 1 µg/ml

of Ab104. The experiments were repeated four times with similar results. B, PP4 co-

purifies with centrosomes. Centrosomes were prepared from 6 x 107 HeLa cells and

purified on a discontinuous sucrose gradient. 10% of protein recovered from each fraction

and 5 µg of HeLa whole cell lysate (W) were Western blotted for the presence of PP4

(Ab 6101) and subcellular compartment markers: γ-tubulin (centrosome), aldolase

(cytosol), lamin B1 (nucleus), GRP78 (ER), and Golgin-97 (golgi), and Bcl-XL

(mitochondria). C, PP4 is a component of the centrosome. HeLa cells were grown on

poly-lysine coated coverslips, extracted in 0.5% Triton X-100 for 2 min and fixed in 4%

ultrapure formaldehyde. Fixed cells were incubated with DAPI DNA stain (DAPI; blue),

human autoimmune serum #4171 (PCM; red), and the peptide-purified anti-PP4

antibody Ab104 (PP4; green; panels a-d) or normal preimmune serum from the same

rabbit used to generate Ab104, before immunization with peptide (n.s.; green; panels e-

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h). Panels PCM, PP4 and DAPI were merged (merged; panel d), to identify areas of

colocalization of PP4 and PCM staining (yellow). Arrows indicate position of

centrosomes. The experiments were repeated at least three times with similar results.

FIG. 2. TNF-α activates both PP4 and JNK in HEK293T cells. A, establishment of PP4

phosphatase assays. 800 µg of HEK293 cell lysate was immunoprecipitated with either

anti-PP4 antibody (Ab104) or protein A bead alone. The immunoprecipitates were

washed and incubated with assay buffer and KTpIRR peptide at 30ºC for various times,

from 0 to 120 min, as indicated (upper panel). 200, 400, 600 or 800 µg of HEK293 cell

lysate was immunoprecipitated with either anti-PP4 antibody (Ab104) or beads alone.

The immunoprecipitates were washed and incubated with assay buffer and KTpIRR

peptide at 30ºC for 30 min (lower panel). The phosphatase assays were read at 650 nm.

The readings are the average and standard deviation of three separate

immunoprecipitations (PP4) or two separate immunoprecipitations (beads). B, TNF-α

activates PP4 phosphatase activity. HEK293T cells were seeded at a density of 3.5x106

per 100 mm dish. After 24 h, the cells were treated with TNF-α (10 ng/ml) for various

times as indicated. PP4 was immunoprecipitated with an anti-PP4 antibody (Ab104). The

PP4 phosphatase activity was determined by using a synthetic peptide, KTpIRR, as a

substrate (upper panel). The amounts of PP4 immunoprecipitated were monitored by

Western blotting using an anti-PP4 antibody (Ab6101; lower panel). The experiments

were repeated at least three times with similar results. C, TNF-α activates JNK kinase

activity. HEK293T cells were seeded at a density of 3.5x106 per 100 mm dish. After 24

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h, the cells were treated with TNF-α (10 ng/ml) for various times as indicated. JNK was

immunoprecipitated with an anti-JNK antibody (Ab101). The JNK phosphatase activity

was determined by using GST-cJun (1-79) as a substrate. The experiments were

repeated three times with similar results.

FIG. 3. TNF-α induces serine and threonine phosphorylation of PP4. A, TNF-α induces PP4

phosphorylation. HEK293T cells (1x 106 cells in 100 mm dish) were transfected with 5 µg Flag-

PP4. After 40 h, the cells were labeled in the phosphate-free DMEM medium supplemented with

5% of dialyzed serum and 100 µCi [32P]orthophosphate/ml for 4 h at 37oC and treated with

TNF-α (10 ng/ml) for the period of time indicated. Flag-PP4 was immunoprecipitated with an

anti-Flag antibody (M2) and subjected to SDS-PAGE. The separated proteins were transferred

to PVDF, and autoradiography was performed. B, TNF-α-induced phosphorylation occurs on

serine and threonine residues of PP4. The corresponding PP4 bands were cut from the PVDF

membrane (A) and subjected to phosphoamino acid analysis. The experiments were repeated at

least two times with similar results.

FIG. 4. JNK activation by TNF-α is blocked by a phosphatase-dead PP4 mutant,

PP4-RL. A, PP4-RL blocks TNF-α-induced JNK activation. HEK293T cells (1.5 x 105

cells in 35 mm wells) were transfected with HA-JNK (0.1 µg) alone or HA-JNK plus

PP4-RL. Empty vector was used to normalize the amount of transfected DNA. 36 h

post-transfection, the cells were treated with TNF-α (10 ng/ml) for 10 min. Cell lysates

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were prepared, HA-JNK1 was immunoprecipitated with an anti-HA antibody (12CA5),

and immunocomplex kinase assays were performed using GST-cJun (1-79) as a

substrate (top panel). Expression levels of HA-JNK and HA-PP4-RL were monitored

by immunoblotting using an anti-HA antibody (12CA5, lower panel). The experiments

were repeated three times with similar results. B, PP4-RL is a phosphatase-dead mutant.

HEK293T cells (1.5 x 106 cells in 100 mm dishes) were transfected with 10 µg of either

PP4 or HA-PP4-RL. The cells were collected 48 h after transfection. PP4 and HA-PP4-

RL were immunoprecipitated with an anti-PP4 (Ab104) and an anti-HA (12CA5)

antibody, respectively. The immunoprecipitates were then subjected to phosphatase

assays (upper panel). The amounts of immunoprecipitated PP4 and HA-PP4-RL were

monitored by Western blotting using an anti-PP4 antibody (Ab6101; lower panel). The

experiments were repeated at least five times with similar results. C, TNF-α-induced

JNK activation was inhibited in HEK293-PP4-RL cells. Parental HEK293 and

HEK293-PP4-RL cells were seeded at a density of 4.5 x 106 cells in 100 mm dishes.

After 24 h, the cells were treated with TNF-α (10 ng/ml) for various times. Cell lysates

were prepared, JNK1 was immunoprecipitated with an anti-JNK1 antibody (Ab101), and

immunocomplex kinase assays were performed using GST-cJun (1-79) as a substrate

(left panel). Cell lysates from parental HEK293 cells and a HA-PP4-RL stably

transfected clone, HEK293-PP4-RL, were subjected to SDS-PAGE and Western

blotting with an anti-PP4 antibody (Ab104) or an anti-HA antibody (12CA5, right

panel).

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FIG. 5. PP4 activates JNK in transient transfection assay. HEK293T cells (1.5 x 105

cells in 35 mm wells) were transfected with HA-JNK1 (0.5 µg) alone or with 2 µg of

PP4, PP1, or Myc-M3/6. Empty vector was used to normalize the amount of transfected

DNA. 36 h post-transfection, the cell lysates were prepared. HA-JNK1 was

immunoprecipitated with an anti-HA antibody (12CA5), and immunocomplex kinase

assays were performed using GST-cJun (1-79) as a substrate. Expression levels of HA-

JNK1, PP4, PP1, and Myc-M3/6 were monitored by immunoblotting using anti-HA

(12CA5), anti-PP4 (Ab104), anti-PP1, and anti-Myc antibodies, respectively (bottom

panels). The experiments were repeated at least ten times with similar results..

FIG. 6. PP4 specifically activates JNK, but not p38 and ERK2. A, HEK293T cells (1.5 x 105

cells in 35 mm wells) were transfected with HA-JNK1 (0.5 µg) alone or HA-JNK1 plus various

amounts of PP4 as indicated. Empty vector was used to normalize the amount of transfected

DNA. 36 h post-transfection, the cell lysates were prepared. HA-JNK1 was immunoprecipitated

with an anti-HA antibody (12CA5), and immunocomplex kinase assays were performed using

GST-cJun (1-79) as a substrate. Expression levels of HA-JNK1 and PP4 were monitored by

immunoblotting using anti-HA (12CA5) and anti-PP4 antibodies, respectively (bottom panels).

The experiments were repeated at least ten times with similar results. B, HEK293T cells (1.5 x

105 cells in 35 mm wells) were transfected with HA-p38 (1 µg) alone, HA-p38 plus various

amounts of PP4, or HA-p38 plus 2 µg of HA-MKK6, as indicated. Empty vector was used to

normalize the amount of transfected DNA. 36 h post-transfection, the cell lysates were prepared.

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HA-p38 was precipitated with an anti-HA antibody (12CA5), and immunocomplex kinase

assays were performed using GST-ATF2 (1-96) as a substrate. Expression levels of HA-p38,

HA-MKK6, and PP4 were monitored by immunoblotting using anti-HA (12CA5) and anti-PP4

antibodies, respectively (bottom panels). C, HEK293T cells (1.5 x 105 cells in 35 mm wells)

were transfected with HA-ERK2 (1 µg) alone, HA-ERK2 plus various amounts of PP4, or HA-

ERK2 plus 1 µg of HA-PKC-ζ, as indicated. Empty vector was used to normalize the amount of

transfected DNA. 36 h post-transfection, the cell lysates were prepared. HA-ERK2 was

precipitated with an anti-HA antibody (12CA5), and immunocomplex kinase assays were

performed using MBP as a substrate. Expression levels of HA-ERK2, HA-PKC-ζ, and PP4

were monitored by immunoblotting using anti-HA (12CA5) and anti-PP4 (Ab104) antibodies,

respectively (bottom panels).

FIG. 7. PP4 does not interact with JNK in vitro. A, no PP4-JNK association was detected in

vitro. HEK293 cells stably transfected with Flag-PP4 (10F1 clone) were seeded at 4x 106 cells per

100 mm dish and treated with TNF-α (10 ng/ml) for 5 min. 600 µg of lysate from either TNF-α

treated or untreated 10F1 HEK293 cells was incubated with GST or GST-JNK fusion protein

immobilized onto glutathione-agarose beads for 2 h at 4oC. The PP4-JNK interaction was

analyzed by immunoblotting with an anti-Flag antibody (M2) to detect Flag-PP4 bound to

GST-JNK after SDS-PAGE (upper panel). The GST and GST-JNK were monitored by

immunoblotting with an anti-GST antibody (middle panel). The experiments were repeated three

times with similar results. To assure Flag-PP4 was in a phosphorylated state, 10F1 HEK293

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cells were labeled in the phosphate-free DMEM medium supplemented with 5% of dialyzed

serum and 100 µCi [32P]orthophosphate/ml for 4 h at 37oC and treated with TNF-α (10 ng/ml)

for 5 min. Flag-PP4 was immunoprecipitated with an anti-Flag antibody (M2) and subjected to

SDS-PAGE and autoradiography. The experiments were repeated two times with similar results.

B, M3/6 associates with JNK in vitro. GST or GST-JNK fusion protein was immobilized on

glutathione-agarose beads and incubated with 600 µg of lysate from HEK293T cells transiently

transfected with Myc-M3/6 for 2 h at 4oC. The M3/6-JNK interaction was analyzed by

immunoblotting with an anti-Myc antibody to detect Myc-M3/6 bound to GST-SAPK after

SDS-PAGE (upper panel). The GST and GST-SAPK were monitored by immunoblotting with

an anti-GST antibody (lower panel).

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Van Hooser, Mickey C.-T. Hu, B. R. Brinkley and Tse-Hua TanGuisheng Zhou, Kathie A. Mihindukulasuriya, Rebecca A. MacCorkle-Chosnek, Aaron

c-Jun N-terminal kinase-induced activation ofαProtein phosphatase 4 is involved in tumor necrosis factor-

published online November 6, 2001J. Biol. Chem. 

  10.1074/jbc.M107014200Access the most updated version of this article at doi:

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