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Originally published In Press as doi:10.1074/jbc.M408067200 on August 24, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46588-46594, November 5, 2004
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Protein Phosphatase 4 Interacts with and Down-regulates Insulin Receptor Substrate 4 following Tumor Necrosis Factor-{alpha} Stimulation*

Kathie A. Mihindukulasuriya{ddagger}§, Guisheng Zhou{ddagger}§, Jun Qin||, and Tse-Hua Tan{ddagger}**

From the {ddagger}Department of Immunology and ||Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030

Received for publication, July 16, 2004 , and in revised form, August 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein phosphatase 4 (PP4; also named PPX or PPP4) is a PP2A-related protein serine/threonine phosphatase with important roles in a variety of cellular processes such as microtubule growth/organization, apoptosis, tumor necrosis factor (TNF)-{alpha} signaling, and activation of c-Jun N-terminal kinase and NF-{kappa}B. To further investigate the cellular functions of PP4, we isolated and identified PP4-interacting proteins using a proteomic approach. We found that insulin receptor substrate 4 (IRS-4) interacted with PP4 and that this interaction was enhanced following TNF-{alpha} stimulation. We also found that PP4, but not PP2A, down-regulated IRS-4 in a phosphatase activity-dependent manner. Pulse-chase analysis revealed that PP4 decreased the half-life of IRS-4 from 4 to 1 h. Moreover, we found that TNF-{alpha} stimulated a PP4-dependent degradation of IRS-4, as indicated by the blockage of the degradation by a potent PP4 inhibitor (okadaic acid) and a phosphatase-dead PP4 mutant (PP4-RL). Taken together, our studies indicate that IRS-4 is subject to regulation by TNF-{alpha} and that PP4 mediates TNF-{alpha}-induced degradation of IRS-4.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin receptor substrate (IRS)1 proteins play a central role in signal transduction by the receptors for insulin, insulin-like growth factor 1 (IGF-1), and a growing number of cytokines and integrins (1). Four IRS proteins have been identified and characterized, including IRS-1, IRS-2, IRS-3 and IRS-4 (2–5). Recently, two more signaling proteins, IRS5/DOK4 and IRS6/DOK5, have been identified as potentially new members of the IRS family (6). IRSs are tyrosine-phosphorylated following insulin or IGF-1 stimulation and form signaling complexes at the receptor with Src homology 2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase, Grb2, Nck, and Shc (7, 8). All IRS proteins share a number of structural and functional characteristics, including an N-terminal pleckstrin homology and a phosphotyrosine binding domain, followed by a large C-terminal region containing multiple sites of tyrosine phosphorylation, which serve as docking sites for Src homology 2 domain-containing signaling proteins. IRS proteins also carry a large number of potential sites for serine and threonine phosphorylation, which may regulate protein-protein interactions (9). Despite the overall similarity, the IRS proteins differ in their subcellular distribution and tissue/developmental expression pattern. Each IRS is phosphorylated on a set of specific tyrosine residues, leading to its binding to the Src homology 2 domains of distinct signaling proteins and therefore, activation of specific signaling cascades. Disruption of each individual IRS gene causes distinct phenotypes in mice (10–14), further indicating that the four IRS proteins play different roles in the regulation of the pleiotropic effects of insulin, IGF-1, and other growth factors.

IRS-4 was initially detected in human embryonic kidney 293 (HEK293) cells (5, 15). Like other IRSs, IRS-4 enhances insulin- and IGF-1-induced mitogenesis in a variety of cells such as HEK293 cells (16, 17), NIH3T3 cells (18), adipose cells (19), and hematopoietic cells (20). It has also been shown that IRS-4 mediates mitogenic signaling by interleukin-4 in hematopoietic cells (20), by growth hormone in LB cells, a murine T-cell lymphoma devoid of IGF-I receptor (21), and by hepatocyte growth factor in pancreatic {beta}-cells (22). IRS-4 has been implicated in liver regeneration as indicated by the substantial induction after partial hepatectomy (23). Recently, it was found that IRS-4 expression level is decreased in polycystic ovary syndrome (PCOS) theca cells (24). IRS-4 has a compensatory role for IRS-1 in adipocyte differentiation (25) and for IRS-2 in pancreatic {beta}-cells (26). However, the negative regulation of IRS-1 and IRS-2 by IRS-4 in IGF-1 signaling has also been suggested (27). IRS-4 lacks both putative SHP-2 binding motifs present in IRS-1, IRS-2, and IRS-3 (15). In comparison with other IRSs, IRS-4 exhibits a more limited tissue expression. Besides HEK293 cells, the IRS-4 protein has only been detected in heart and skeletal muscle cells (28), although IRS-4 mRNA is expressed in a variety of human and rodent tissues including pituitary, thyroid, ovary, prostate, hypothalamus, liver, heart, and skeletal muscle (29). Tissue-specific expression of IRS-4 suggests the potential involvement of IRS-4 in the regulation of insulin/IGF-1 signaling in these tissues. Five amino acid polymorphisms have been identified in IRS-4, although their functional relevance remains to be determined (30). To date, only a few IRS-4-interacting proteins have been identified, including the regulatory and catalytic subunits of phosphatidylinositol 3-kinase (23, 31), the imidazoline receptor antisera selected (31), the suppressor of cytokine signaling-6 (32), Src homology phosphatase (23), and protein kinase C-{zeta} (23). Identification and characterization of novel IRS-4-interacting proteins will help in understanding the cellular functions of IRS-4.

Protein phosphatase 4 (PP4; previously called PPX) (33) is a member of the PP2A subfamily of protein serine/threonine phosphatases, along with PP2A and PP6 (34). PP4, PP6, and PP2A are highly homologous; human PP4 shares 65% identity with PP2A (33, 35). PP4, like PP2A, is a holoenzyme composed of catalytic (C), structural (A), and regulatory (B) subunits. To date, three subunits have been identified for PP4: {alpha}4, PP4-R1, and PP4-R2 (36–40). Like PP2A, PP4 contains a putative binding domain for okadaic acid, a potent tumor promoter toxin (41). Okadaic acid inhibits PP4 with a similar range of concentration (IC50 = 0.1 nM in vitro) as PP2A (42). PP4 is involved in the regulation of microtubule growth or organization at the centrosomes (43) and the centrosome maturation in mitosis and meiosis (44). It has been recently shown that PP4 plays an important proapoptotic role in T lymphocytes (45). Our previous studies found that PP4 interacts with members of the NF-{kappa}B family of transcription factors c-Rel and RelA and activates NF-{kappa}B-mediated transcription (46). Recent studies have demonstrated that PP4 dephosphorylates RelA (also called NF-{kappa}B p65), primarily on Thr435, and that this dephosphorylation is required for NF-{kappa}B activation induced by cisplatin and extracellular signal-regulated kinase suppression (47). We have also found that PP4 plays a role as a positive regulator of the c-Jun N-terminal kinase pathway in TNF-{alpha} signaling (48). Recently, PP4 has been found to interact with the survival of motor neurons complex and enhances the temporal localization of small nuclear ribonucleoproteins (49). Given the high degree of conservation of PP4 during evolution (33, 35, 50), it is likely that PP4 may participate in many essential cellular processes.

In this study, by applying a functional proteomic approach, we identified IRS-4 as a novel PP4-interacting protein. Moreover, we found that the stability of IRS-4 was subject to regulation by TNF-{alpha} and that PP4 mediated the degradation of IRS-4 by TNF-{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—The SuperSignal chemiluminescence system was purchased from Pierce. TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). The mixture of [35S]methionine-cysteine was purchased from ICN Biomedicals (Irvine, CA). Anti-HA antibody (12CA5) was purchased from Roche Applied Science. Anti-FLAG (M2) and anti-{gamma}-tubulin antibodies were purchased from Sigma. Anti-{alpha}4 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-IRS-4 antibody (Ab 1640), anti-PP4-R2 antibody (Ab 1641), and anti-PP2A antibody (Ab 1643) were raised against the C-terminal regions of IRS-4 (CDFARRDNQFDSPKRGR1242–1257), PP4-R2 (CESFMT-SREMIPERK388–401), and PP2A (CDTHPRGYGNRQNMG326–339), respectively. Anti-PP4-R1 antibody (Ab 1650) was raised against the N-terminal region of PP4-R1 (CASENIFNRQMVARS39–52). Rabbit anti-PP4 polyclonal antibodies (Ab 104 and 6101) were previously described (48). Ab 1640 (anti-IRS-4) and Ab 6101 (anti-PP4) were peptide-purified using the Sulfolink Kit from Pierce. The Sepharose beads were purchased from Amersham Biosciences. All other chemical reagents were purchased from Sigma unless otherwise noted.

Plasmids—pEF-HA-IRS-4 was provided by Dr. D. LeRoith (National Institutes of Health, Bethesda, MD) (18). pBJF-FLAG-PP2A was kindly provided by Dr. J. Chen (University of Illinois at Urbana-Champaign) (37). pCI-neo-FLAG-PP4 (48), PP4, and HA-PP4-RL (46) were previously described.

Cells and Transfection—HEK293 and HEK293T cells were grown and maintained as previously described (48). HEK293 and HEK293T cells were plated at a density of either 2.5 x 105 cells/35-mm plate well or 1.6 x 106 cells/100-mm dish and transfected the next day using the modified calcium phosphate precipitation protocol (Specialty Media, Inc., Lavallette, NJ). Cells were lysed in 1% Nonidet P-40 lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40) freshly supplemented with 6.6 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM dithiothreitol, 50 µM p-amidinophenylmethanesulfonyl fluoride, 50 mM NaF, and 1 mM Na3VO4.

Co-immunoprecipitation and Western Blot Analysis—Co-immunoprecipitation assays were performed as previously described (51), except that the immunoprecipitates were washed three times with ice-cold NETN buffer (20 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Western blot analysis was performed using an enhanced chemiluminescence detection kit according to the manufacturer's protocols (Pierce).

Phosphatase Assay—HEK293T 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-amidinophenylmethanesulfonyl fluoride, and 2 µg/ml aprotinin. PP4 was immunoprecipitated with an anti-PP4 (Ab 104) antibody. FLAG-PP2A was immunoprecipitated with anti-FLAG (M2) antibody. 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). In brief, 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 30 °Cfor 30 min. 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/ml 10% Tween 20 added fresh). After 15 min at room temperature, the assay was read at 650 nm on a PerkinElmer bioassay reader (HTS 7000 plus).

Establishment of the HEK293 Cell Clone (10F1) Stably Transfected with FLAG-PP4—HEK293 cells were grown in complete Dulbecco's modified Eagle's medium containing 10% fetal calf serum supplemented with 12.5 mM HEPES, 50 µg/ml gentamycin, and 100 units/ml penicillin/streptomycin (Invitrogen). We transfected the HEK293 cells with FLAG-PP4 by the Fugene 6 method according to the manufacturer's protocol (Roche Applied Science). The transfected cells were selected by Geneticin (G418; Invitrogen) at a concentration of 750 µg/ml. The cells were replated at a 1:15 dilution whenever they reached 80% confluence. After 10–14 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.

Isolation of FLAG-PP4-interacting Protein Complexes—HEK293 cells stably expressing FLAG-PP4, designated as 10F1 clone, were left unstimulated or stimulated with TNF-{alpha} (10 ng/ml) for 10 min. Fifteen confluent T175 flasks of 10F1 or parental HEK293 cells (5.175 x 108 cells) were used per purification. The cells were trypsinized, washed twice with ice-cold phosphate-buffered saline, and stored at –80 °C until needed. The cells were lysed in 5 ml of PIP lysis buffer (20 mM HEPES (pH 7.4), 2 mM EDTA, 1% Triton X-100, 150 mM NaCl) freshly supplemented with 6.6 µg/ml aprotinin, 10 µg/ml leupeptin, and 50 µM p-amidinophenylmethanesulfonyl fluoride. The lysate was centrifuged at 20,817 x g (14,000 rpm) for 15 min to remove cellular debris. Chromosomal DNA was sheared with a 1-ml syringe. The whole cell extract (105 mg) was incubated with either anti-FLAG-Sepharose or unconjugated Sepharose at 4 °C for 3 h with continuous rotation, packed into a column, and washed three times with 10 ml of NETN buffer. FLAG-PP4 was eluted four times with 1x column volume of 100 µg/ml FLAG peptide in TBS (50 mM Tris-HCl (pH 7.4) and 150 mM NaCl). 0.1 µl of each 100-µl fraction was used for anti-FLAG Western blotting to determine which fraction(s) contained FLAG-PP4. The fraction containing the most FLAG-PP4 and the corresponding fractions for the negative controls were separated by 10% SDS-PAGE, and the gel was stained with Coomassie Blue.

Identification of Proteins by Mass Spectrometry—Identification of proteins by mass spectrometry was performed as previously described (52, 53). A MALDI-TOF mass spectrometer with delayed extraction, a home-built MALDI-ion trap mass spectrometer, and an electrospray ion trap mass spectrometer (LCQ; Finnigan MAT, San Jose, CA) coupled on-line with a capillary high pressure liquid chromatograph (Magic 2002; Michom BioResources, Auburn, CA) were used to acquire tandem mass spectra. A 0.1 x 50-mm Magic MS C18 column (5-µm particle diameter, 200-Å pore size) with mobile phases A (methanol/water/acetic acid, 5:95:1) and B (methanol/water/acetic acid, 85:15:1) was used with a gradient of 2–98% mobile phase B over 2.5 min, followed by 98% mobile phase B for 2 min. Specific protein bands from Coomassie Blue-stained SDS-polyacrylamide gels were excised, destained, and digested with trypsin (200 ng/digestion) in 20 µl of 50 mM NH4HCO3 buffer for 2 h at 37 °C using a protein/enzyme weight ratio of 1:1. The resulting peptides were extracted, and 20–50% of the sample was used to obtain liquid chromatography/tandem mass spectra. The tandem mass spectra were used to search the compiled NCBI nonredundant protein and expressed sequence tag data bases with the program Pep-Frag to identify the proteins.

Pulse-chase Analysis—HEK293T cells were plated at a density of 1.5 x 105 cells per 35-mm plate well and transfected the next day with HA-IRS-4 (2 µg) plus empty vector or PP4 (2 µg) as described above. Thirty-six h after transfection, the medium was replaced with 2 ml of prelabeling medium (Dulbecco's modified Eagle's medium lacking cysteine and methionine + 5% fetal calf serum). After 1 h of incubation, the prelabeling medium was replaced with 1 ml of labeling medium (prelabeling medium + 0.1 mCi/ml [35S]methionine and [35S]cysteine). The cells were labeled for 4 h and then chased with fresh, nonradioactive medium for the times indicated. The cells were lysed in lysis buffer (20 mM HEPES (pH 7.4), 2 mM EDTA, 1% Triton X-100, 10% glycerol, and 150 mM NaCl) freshly supplemented with 6.6 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM dithiothreitol, and 50 µM p-amidinophenylmethanesulfonyl fluoride. 100 µg of cell lysate was immunoprecipitated with an anti-HA antibody (12CA5) and protein A-Sepharose and resolved by SDS-PAGE. The gels were dried and autoradiographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IRS-4 Is a TNF-{alpha}-inducible, PP4-interacting Protein—We have previously shown that PP4 is involved in TNF-{alpha} signaling (48). In the search for novel cellular functions of PP4, we established a HEK293 cell clone (10F1) stably transfected with FLAG-PP4 and applied a functional proteomic approach to identify TNF-{alpha}-inducible, PP4-interacting protein(s) in 10F1 cells. We treated 10F1 cells with TNF-{alpha}, and the whole cell extract of 10F1 cells was incubated with anti-FLAG-Sepharose. After extensive washing, FLAG-PP4 and the proteins that complexed with PP4 were eluted with FLAG peptide, subjected to SDS-PAGE, and stained with Coomassie Blue (Fig. 1A). By mass spectrometry, a group of potential PP4-interacting proteins in response to TNF-{alpha} stimulation was identified (Fig. 1A). We selected one of these proteins, insulin receptor protein 4 (IRS-4), a docking protein that coordinates cellular responses to insulin and insulin-like growth factor 1 (IGF-1), for further study. A representative spectrum is shown for IRS-4 along with the peptide sequence and its position in the IRS-4 protein (Fig. 1B). To confirm the interaction between PP4 and IRS-4, we took a fraction of the FLAG peptide elution and performed Western blot analysis using an anti-IRS-4 antibody. As shown in Fig. 2, the presence of IRS-4 in FLAG-PP4-interacting protein complex was greatly increased upon TNF-{alpha} treatment (top panel). These data indicate that IRS-4 is a TNF-{alpha}-inducible, PP4-interacting protein. We also found that three known PP4-interacting proteins, {alpha}4, PP4-R1, and PP4-R2, co-purified with FLAG-PP4 (Fig. 2), further confirming the binding specificity of our approach.



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  		FIG. 1.
Isolation and identification of IRS-4 as a TNF-{alpha}-inducible PP4-interacting protein. A, isolation of PP4-interacting proteins. Whole cell extracts from 5 x 108 HEK293 cells stably expressing FLAG-PP4 (10F1 clone), unstimulated (lane 3), or stimulated for 10 min with 10 ng/ml TNF-{alpha} (lane 4) or the parental HEK293 cells (lane 2) were incubated with anti-FLAG-Sepharose for 2 h at 4 °C. As an additional control, 10F1 cells were incubated with unconjugated Sepharose (lane 1). The Sepharose was then placed in a column and washed three times with NETN buffer. FLAG-PP4 and the PP4-interacting proteins were eluted four times with one-column volume of 100 µg/ml FLAG peptide. Twelve fractions(30 µl each) were collected, and the fraction containing the most FLAG-PP4, as determined by Western blotting, was subjected to SDS-PAGE and stained with Coomassie Blue. Several protein bands, which appeared after TNF-{alpha} stimulation (lane 4), were sequenced via a MALDI-TOF mass spectrometer and identified as indicated. B, mass spectrometry analysis and identification of IRS-4. A representative tandem mass spectrometry spectrum that identifies the 160-kDa band isolated from TNF-{alpha}-stimulated cells as the novel PP4-interacting protein, IRS-4.

 



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  		FIG. 2.
IRS-4 co-purifies with PP4. Equal amounts of eluate from the fractionation described in the legend to Fig. 1A were separated via SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were Western blotted with antibodies to IRS-4, {alpha}4, PP4-R1, PP4-R2, and PP4 (FLAG).

 
To further confirm the PP4-IRS-4 interaction, we transiently transfected 10F1 cells with HA-IRS-4, immunoprecipitated FLAG-PP4, and performed Western blotting against HA-IRS-4. We found that FLAG-PP4 interacts with HA-IRS-4 and that this interaction was greatly enhanced by TNF-{alpha} stimulation (Fig. 3A). To exclude the possibility of an artificial interaction due to the overexpression system and to demonstrate the physiologic relevance of this TNF-{alpha}-inducible interaction, we performed immunoprecipitation/Western blot analysis to determine whether endogenous PP4 and IRS-4 interact with each other in HEK293 cells. We treated HEK293 cells with TNF-{alpha}, immunoprecipitated endogenous PP4, and Western blotted against endogenous IRS-4. As with the overexpressed proteins, endogenous PP4 and IRS-4 interacted with each other, and this interaction was enhanced after TNF-{alpha} stimulation (Fig. 3B). Taken together, our data indicate that IRS-4 is a PP4-interacting protein and that the PP4-IRS-4 interaction is subject to regulation by TNF-{alpha} stimulation.



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  		FIG. 3.
TNF-{alpha} enhances the PP4-IRS-4 interaction. A, 10F1 cells were transfected with equal amounts of empty vector or HA-IRS-4. Twenty-four h after transfection, the cells were left unstimulated or stimulated with TNF-{alpha} (10 ng/ml) for 2.5 min. FLAG-PP4 was immunoprecipitated with an anti-FLAG antibody (M2). The PP4-IRS-4 interaction was detected by immunoblotting with an anti-HA antibody (top). Levels of FLAG-PP4 and HA-IRS-4 in the lysates were monitored by immunoblotting lysate with anti-FLAG (bottom) and anti-IRS-4 (middle) antibodies, respectively. B, HEK293 cells were left unstimulated or stimulated with TNF-{alpha} (10 ng/ml) for 2.5 min. The lysates were immunoprecipitated with an anti-PP4 antibody. The PP4-IRS-4 interaction was detected by immunoblotting with a peptide-purified anti-IRS-4 antibody (top). Equal immunoprecipitation of PP4 was monitored by immunopreciptating with an anti-PP4 antibody, followed by immunoblotting with a peptide-purified anti-PP4 antibody (bottom).

 
PP4 Down-regulates IRS-4—To determine the functional relevance of the PP4-IRS-4 interaction, we examined the effect of PP4 on the protein level of IRS-4. We co-transfected HA-IRS-4 into HEK293 cells with increasing amounts of PP4 or PP2A, the most closely related serine/threonine phosphatase. Western blot analysis showed that co-transfection of PP4 resulted in down-regulation of IRS-4 in a dose-dependent manner, whereas PP2A had no such an effect (Fig. 4A). Our phosphatase assays demonstrated that overexpressed PP4 and PP2A had comparable phosphatase activity under these experimental conditions (Fig. 4B). Thus, these data indicate that the function of the PP4-IRS-4 interaction is to down-regulate IRS-4 expression. In order to corroborate the role of PP4 in regulating IRS-4 protein levels, we examined the effect of PP4-RL, a phosphatase-dead mutant of PP4 in which arginine 236 is replaced with leucine (46, 48), on IRS-4. We found that IRS-4 was degraded in the presence of wild-type PP4 but not phosphatase-dead PP4-RL (Fig. 4C). These results indicate that PP4 phosphatase activity is required for its effect on IRS-4.



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  		FIG. 4.
PP4, but not PP2A, down-regulates IRS-4. A, HEK293 cells were transfected with HA-IRS-4 and increasing amounts of PP4 or FLAG-PP2A. Empty vector was used to normalize the amount of transfected DNA. Twenty µg of whole cell lysate was subjected to Western blotting with an anti-HA antibody, anti-PP4 antibody, anti-FLAG antibody, and anti-{alpha}-tubulin antibody, respectively. B, PP4 and PP2A have comparable phosphatase activities. HEK293T cells (1.5 x 106 cells in a 100-mm dish) were transfected with vector alone, PP4 (10 µg), or FLAG-PP2A (10 µg). The cells were collected 40 h post-transfection. PP4 and FLAG-PP2A were immunoprecipitated with anti-PP4 (Ab 104) and anti-FLAG (M2) antibodies, respectively. The immunoprecipitates were then subject to phosphatase assays. C, HEK293 cells were transfected with HA-IRS-4 and equivalent amounts of PP4 or HA-PP4-RL. Empty vector was used to normalize the amount of transfected DNA. Twenty µg of whole cell lysate was subjected to Western blotting with an anti-IRS-4 antibody. An equal amount of lysate was subjected to Western blotting with an anti-PP4 antibody to ensure equal expression of PP4 and HA-PP4-RL and then reprobed with an anti-{gamma}-tubulin antibody to serve as a loading control.

 
PP4 Decreases the Half-life of IRS-4—To determine the underlying mechanism by which PP4 down-regulates IRS-4, we determined the half-life of IRS-4 in the presence or absence of PP4. HEK293T cells were transfected with HA-IRS-4 plus empty vector (Fig. 5A) or PP4 (Fig. 5B), pulsed with a mixture of [35S]methionine and [35S]cysteine, and then chased with nonradioactive medium for various periods of time. The half-life of IRS-4 decreased from 4 h (Fig. 5A) to 1 h in the presence of PP4 (Fig. 5B). Thus, our data indicate that overexpression of PP4 leads to the enhanced degradation of IRS-4 protein.



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  		FIG. 5.
PP4 decreases the half-life of IRS-4. HEK293T cells were transfected with HA-IRS-4 plus empty vector (A) or PP4 (B) and labeled with a mixture of [35S]methionine and [35S]cysteine for 4 h. The cells were chased with fresh, nonradioactive medium for the times indicated. IRS-4 was immunoprecipitated with an anti-HA antibody, resolved by SDS-PAGE, and subjected to autoradiography (top). Densitometric analysis of HA-IRS-4 was done using Kodak 1D Image Analysis Software (bottom). The amount of HA-IRS-4 at chasing time 0 h was set equal to 100%.

 
PP4 Mediates TNF-{alpha}-induced Degradation of IRS-4—We have previously shown that TNF-{alpha} stimulates PP4 phosphatase activity (48). Given the observation that TNF-{alpha} enhanced the PP4-IRS-4 interaction (Figs. 2 and 3), we wondered whether TNF-{alpha} could stimulate the degradation of IRS-4. To this end, we stimulated HEK293 cells with varying amounts of TNF-{alpha} and assayed the lysate for the protein levels of IRS-4. We found that TNF-{alpha} treatment led to the degradation of IRS-4 in a dose-dependent manner (Fig. 6A). To determine whether PP4 was involved in the TNF-{alpha}-induced degradation of IRS-4, we pretreated HEK293 cells with okadaic acid, a potent PP4 and PP2A inhibitor (41), and then treated the cells with TNF-{alpha}. We found that pretreatment with okadaic acid blocked TNF-{alpha}-induced degradation of IRS-4 (Fig. 6B). Okadaic acid inhibits both PP4 and PP2A at nearly the same concentration (42). Therefore, to confirm the specific involvement of PP4 in TNF-{alpha}-induced degradation of IRS-4, we transfected HEK293T cells with HA-IRS-4 plus empty vector or the phosphatase-dead mutant, HA-PP4-RL, and left the cells unstimulated or stimulated with TNF-{alpha}. Western blot analysis indicates that PP4-RL was able to completely block the degradation of IRS-4 by TNF-{alpha} (Fig. 6C). Taken together, the degradation of IRS-4 following TNF-{alpha} stimulation is dependent on the phosphatase activity of PP4.



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  		FIG. 6.
PP4 mediates the TNF-{alpha}-stimulated degradation of IRS-4. A, HEK293 cells were stimulated for 1 h with various concentrations of TNF-{alpha}, as indicated. The cells were lysed, and 20 µg of whole cell lysate was subjected to Western blotting with an anti-IRS-4 antibody and then reprobed with an anti-{gamma}-tubulin antibody to serve as a loading control. B, HEK293 cells were either untreated or pretreated with 50 nM okadaic acid for 4 h and then treated with 50 ng/ml TNF-{alpha} for 1 h. The cells were lysed, and 20 µg of whole cell lysate was subjected to Western blotting with an anti-IRS-4 antibody and then reprobed with an anti-{gamma}-tubulin antibody to serve as a loading control. C, HEK293T cells were transfected with HA-IRS-4 plus empty vector or HA-PP4-RL and then left untreated or treated with 100 ng/ml TNF-{alpha} for 2 h. The cells were lysed, and 20 µg of whole cell lysate was subjected to Western blotting with an anti-IRS-4 antibody and then reprobed with anti-HA to show the expression of HA-PP4-RL and with an anti-{gamma}-tubulin antibody to serve as a loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PP4 is a highly conserved, PP2A-related serine/threonine phosphatase. To date, the cellular functions of PP4 and the underlying PP4-mediated signal transduction mechanisms are largely unknown. Moreover, okadaic acid, a derivative of a dinoflagellate toxin (41), inhibits PP4 with an IC50 of about 0.1 nM, comparable with that of PP2A (42). It is likely that some cellular functions previously assigned to PP2A may actually belong to PP4. We have previously identified both the c-Jun N-terminal kinase and NF-{kappa}B pathways as potential PP4-targeted signaling pathways (46, 48). Here, we provide both biochemical and pharmacological evidence that PP4 is involved in the regulation of IRS-4 and the IRS-4-mediated signaling pathway(s). By functional proteomic analysis, we identified IRS-4 as a TNF-{alpha}-inducible, PP4-interacting protein. Moreover, we found that the interaction of PP4 with IRS-4 led to the degradation of IRS-4 through a decrease in the half-life of IRS-4. The physiological relevance of the PP4-IRS-4 interaction was shown by the complete blockage of TNF-{alpha}-induced degradation of IRS-4 by okadaic acid and by a dominant-negative PP4 mutant. Taken together, our study identifies the IRS-4-mediated signaling pathway(s) as another novel PP4-targeted pathway.

It has been shown that TNF-{alpha} plays an inhibitory role in insulin signaling and contributes to the development of insulin resistance (54). Multiple mechanisms, not mutually exclusive, have been proposed to account for TNF-{alpha}-induced insulin resistance in obesity, including the elevation of plasma free fatty acids due to its lipolytic action, the down-regulation of the translocation of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane, the antagonism of the peroxisome proliferator-activated receptor {gamma} pathway, and the activation of c-Jun N-terminal kinase and inhibitor {kappa}B kinase (55–58). TNF-{alpha} has also been shown to directly interfere with the early steps of insulin signaling. It has been shown that IRS-1 and IRS-2 are prominent TNF-{alpha} targets within the insulin signaling cascade and major integration points of the TNF-{alpha} and insulin signaling pathways. For example, TNF-{alpha} treatment increases the serine/threonine phosphorylation of IRS-1 and IRS-2, which in turn leads to their dissociation from the insulin receptor (55, 59, 60), the down-regulation of IRS proteins (61–63), and thus the inhibition of insulin signaling. We provide evidence here for the first time that IRS-4 is degraded in response to TNF-{alpha} stimulation. Since IRS-4 has been shown to be involved in many aspects of insulin signaling, our studies reveal a novel mechanism by which TNF-{alpha} regulates IRS-4 and IRS-4-mediated insulin signaling.

TNF-{alpha} also inhibits the signaling of another IRS-mediated pathway, the IGF-1 pathway. For example, TNF-{alpha} opposes the antiapoptotic function of IGF-1 signaling in neurons and breast cancer cells (64–66). Like other IRS family proteins, IRS-4 has also been shown to mediate signals from IGF-1 as indicated by its tyrosine phosphorylation after IGF-1 stimulation (28). Recently, PP4 has been shown to be a proapoptotic protein (45). Thus, it is likely that PP4 may play a role in TNF-{alpha}-induced inhibition of the antiapoptotic function of IGF-1.

Multiple serine phosphorylation sites, targeted by different protein kinases under different conditions, have been identified in IRS-1. Whereas most serine phosphorylation identified in IRS-1 so far inhibits IRS-1-mediated insulin signaling, some serine/threonine phosphorylation of IRS-1 exerts a positive effect on insulin signaling. For example, the phosphorylation of serine residues within the phosphotyrosine binding domain of IRS-1 by insulin-stimulated protein kinase B protects IRS-1 protein from the rapid action of protein-tyrosine phosphatases and enables the Ser-phosphorylated IRS-1 protein to maintain its Tyr-phosphorylated, active conformation (67). Recently, phosphorylation of rat IRS-1 on Ser-302 was found to be required for insulin-stimulated tyrosine phosphorylation of IRS-1 and the IRS-1-phosphatidylinositol 3-kinase interaction (68). Moreover, some basal serine phosphorylation of IRS-1 is necessary for insulin signaling (69). However, the serine/threonine phosphorylation site(s) responsible for the stabilization of IRS proteins has not been identified yet. Given the high homology between IRS-1 and IRS-4, it is likely that IRS-4 is also subject to regulation by serine phosphorylation. Given the fact that PP4 is a serine/threonine phosphatase, combining our observations that PP4 interacted with and down-regulated IRS-4 through decreasing the half-life of IRS-4, we hypothesize that PP4 dephosphorylates a functional serine/threonine residue(s), whose phosphorylation contributes to the stabilization of IRS-4, leading to the enhanced degradation of IRS-4. Therefore, identification of PP4-targeted phosphorylation site(s) will provide new insight into the understanding of the regulation of IRS proteins in vivo.


   FOOTNOTES
 
* This work was supported by National Institutes of Health (NIH) Grant R01-CA87076 (to T.-H. T.), American Heart Association, Texas Affiliate, Beginning Grant-in-aid 0465156Y (to G. Z.), United States Army Breast Cancer Research Program Predoctoral Fellowship DAMD 17-011-0139, and NIH Training Grant T32-AI07495 (to K. A. M.). 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.

This paper is dedicated to the memory of Suzanne Robertson, whose kindness and helpfulness bettered the lives of all of those who had the privilege to work with her. Back

§ These two authors contributed equally to this work. Back

To whom correspondence may be addressed: Dept. of Immunology, Baylor College of Medicine, One Baylor Plaza, M929, Houston, TX 77030. E-mail: guisheng{at}bcm.tmc.edu. ** To whom correspondence may be addressed: Dept. of Immunology, Baylor College of Medicine, One Baylor Plaza, M929, Houston, TX 77030. E-mail: ttan{at}bcm.tmc.edu.

1 The abbreviations used are: IRS, insulin receptor substrate; PP4, protein phosphatase 4; PP2A, protein phosphatase 2A; TNF-{alpha}, tumor necrosis factor-{alpha}; IGF-1, insulin-like growth factor-1; HA, hemagglutinin; HEK293, human embryonic kidney 293; Ab, antibody; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. Back


   ACKNOWLEDGMENTS
 
We thank our colleagues for providing valuable reagents; members of the Tan laboratory for helpful discussions and critical reading of the manuscript; M. Hu for technical assistance; and S. Robertson and Denise Guzman for secretarial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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