RNAi Screen in Mouse Astrocytes Identifies Phosphatases that Regulate NF-κB Signaling

Molecular Cell 24, 497–509, November 17, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2006.10.015

RNAi Screen in Mouse Astrocytes Identifies Phosphatases that Regulate NF-kB Signaling Shitao Li,1 Lingyan Wang,1 Michael A. Berman,1 Ye Zhang,1,2 and Martin E. Dorf1,* 1 Department of Pathology Harvard Medical School Boston, Massachusetts 02115

Summary Regulation of NF-kB activation is controlled by a series of kinases; however, the roles of phosphatases in regulating this pathway are poorly understood. We report a systematic RNAi screen of phosphatases that modulate NF-kB activity. Nineteen of 250 phosphatase genes were identified as regulators of NF-kB signaling in astrocytes. RNAi selectively regulates endogenous chemokine and cytokine expression. Coimmunoprecipitation identified associations of distinct protein phosphatase 2A core or holoenzymes with the IKK, NF-kB, and TRAF2 complexes. Dephosphorylation of these complexes leads to modulation of NF-kB transcriptional activity. In contrast to IKK and NF-kB, TRAF2 phosphorylation has not been well elucidated. We show that the Thr117 residue in TRAF2 is phosphorylated following TNFa stimulation. This phosphorylation process is modulated by PP2A and is required for TRAF2 functional activity. These results provide direct evidence for TNF-induced TRAF2 phosphorylation and demonstrate that phosphorylation is regulated at multiple levels in the NF-kB pathway. Introduction Astrocytes are the most abundant glial cell type in the central nervous system (CNS). They contribute to homeostasis of the CNS by participation in neurogenesis (Song et al., 2002), synapse formation (Mauch et al., 2001), synaptic transmission (Kang et al., 1998), brain repair (Garcia-Segura et al., 1999), and maintenance of the blood-brain barrier (Bush et al., 1999; Prat et al., 2001). Astrocytes also play a role in the pathophysiology of inflammatory and neurodegenerative diseases (Volterra and Meldolesi, 2005). Brain lesions observed in Alzheimer’s disease, ischemic damage, autoimmune responses, infections (e.g., HIV), and tumors are rapidly bordered by hypertrophic astrocytes. These reactive astrocytes can produce a variety of proinflammatory mediators that amplify the inflammatory response. Tumor necrosis factor-a (TNFa) plays a critical role in the induction and perpetuation of innate, immune, and inflammatory responses. TNFa signaling occurs through specific receptors that induce activation of NF-kB along with other transcription factors (MacEwan, 2002). NF-kB plays an essential role in inflammation, immunity, development, cell proliferation, and apoptosis (Hayden and *Correspondence: [email protected] 2 Present address: Department of Biochemistry and Molecular Biology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China, 100005.

Ghosh, 2004). The activity of NF-kB is tightly regulated by association with an inhibitor of NF-kB (IkB). NF-kB bound to IkB is found in the cytoplasm as an inactive complex. However, following TNFa treatment, the IkB kinase (IKK) is activated, resulting in phosphorylation of IkB proteins. This signal-induced phosphorylation targets IkB for polyubiquitination and subsequent degradation, allowing the freed NF-kB molecules to translocate to the nucleus and modulate specific gene transcription. Phosphorylation has been shown to regulate the various steps in NF-kB signaling (Hayden and Ghosh, 2004; Viatour et al., 2005), a process that is controlled by kinases and phosphatases with opposing roles. Dozens of kinases have been demonstrated to be involved in the phosphorylation of IkB, NF-kB, and other components in the NF-kB pathway (Hayden and Ghosh, 2004; Viatour et al., 2005). In contrast to the extensive analysis of kinase function, the roles of phosphatases in NF-kB signaling remain poorly understood. In this study, a large-scale RNAi screen was adopted to elucidate the roles of phosphatases in the NF-kB pathway. After two rounds of screening, 19 phosphatases were identified as regulators of NF-kB signaling either activating or suppressing NF-kB transcriptional activity and binding ability. Distinct protein phosphatase 2A (PP2A) enzymes were associated with the IKK, NFkB, and TRAF2 complexes. Dephosphorylation of these complexes led to inhibition of NF-kB transcriptional activity and regulation of endogenous chemokine or cytokine expression in astrocytes. Results Primary Screen by Using siRNA Library To identify which phosphatases were involved in the NF-kB pathway, a large-scale RNAi approach was adopted to characterize the role of individual phosphatase genes. A siRNA library comprising 250 phosphatase or putative phosphatase genes was prepared based on a bidirectional siRNA vector transcribing siRNAs from convergent opposing promoters (Kaykas and Moon, 2004; Zheng et al., 2004) (Figure 1A). Astrocytes were transfected with the pNF-kB-Luc and Renilla luciferase reporters plus a pair of siRNA constructs for each gene. The Renilla luciferase vector was used as a control of transfection efficiency. TNFa-treated or -untreated astrocytes were used to screen phosphatases involved in NF-kB activation. Genes that satisfied the following four criteria of activity and specificity were categorized as positive candidates. (1) Genes scoring two standard deviations (SD) above or below the median were considered potential hits. Two SD roughly equal a 4-fold increase over media controls, a 3-fold increase over the TNFa-treated control, or a 70% reduction in NF-kB activity as measured by changes of NF-kB reporter activity (Table 1). (2) Candidate genes exhibited reporter specificity by demonstrating <2-fold activity changes with a mutant NF-kB reporter on the same vector backbone. (3) The expression and activity of each phosphatase gene was

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Figure 1. RNAi Screens of Phosphatase Genes that Regulate NF-kB Transcriptional Activity (A) The design of the pBabe-Dual vectors. The positions of the puromycin resistance gene and the U6 and H1 promoters are indicated. (B) Data from representative experiments depict the modulation of NF-kB reporter activity for each of the 250 phosphatase siRNA targets in nonTNF-treated astrocytes. (C) The regulation of NF-kB reporter activity after TNFa stimulation of astrocytes transfected with each pair of siRNA targeting constructs. The luciferase activity of cells transfected with control siRNA vector with TNFa was set at 1. The activity of NF-kB signaling was quantified by measurement of the log ratio of firefly luciferase activity as standardized to that of Renilla luciferase. Dashed lines indicate two standard deviations (2 3 SD). (D) Knockdown of 13 phosphatases activated NF-kB-binding activity in CTFA. White bars represent responses of unstimulated astrocytes; shaded bars represent responses of TNFa-treated cells. Stimulation with TNFa triggered a 6- to 9-fold increase in CTFA activity; this value was normalized to 1 for comparison. Error bars represent the SD of three independent experiments. (E) Knockdown of six phosphatases suppressed NF-kB-binding activity in CTFA assays. Error bars represent the SD of three independent experiments. (F) The fold induction of NF-kB reporter activity after treatment with TNFa (shaded bars) or medium (open bars) in NIH3T3 cells transfected with the indicated pair of phosphatase siRNA targeting constructs.

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Table 1. Identification of Phosphatase Genes Associated with the NF-kB Pathway

GenBank Accession Number

RNAi Reporter Activity Symbol

Name

Basal

TNFa

Phosphatase Specificity

Protein phosphatase 2A, catalytic subunit, alpha isoform Protein phosphatase 2A, catalytic subunit, beta isoform Protein phosphatase 2C epsilon Protein phosphatase 2A, regulatory subunit B, gamma isoform Protein phosphatase 2C zeta Protein phosphatase 2A, regulatory subunit A, beta isoform Protein phosphatase 2A, regulatory subunit B, delta isoform Phosphatase subunit gene g4-1 Protein phosphatase 1, regulatory (inhibitor) subunit 12C Protein tyrosine phosphatase, nonreceptor type 21 Protein phosphatase 2A, regulatory subunit A, alpha isoform Inositol polyphosphate-4-phosphatase, type I Phosphatase and tensin homolog

10.1

5.6

Ser/Thr

8.1

4.1

Ser/Thr

7.7 6.5

4.7 3.9

Ser/Thr Ser/Thr

5.7 5.5

4.5 3.9

Ser/Thr Ser/Thr

5.4

4.4

Ser/Thr

4.5 6.6

3.6 1.2

Ser/Thr Ser/Thr

4.2

0.8

Tyr

4.0

1.8

Ser/Thr

1.9 1.4

5.1 4.1

Lipid Lipid and Tyr

Protein phosphatase 1, regulatory (inhibitor) subunit 7 Protein tyrosine phosphatase, receptor type, N Protein phosphatase 2C delta Protein tyrosine phosphatase, receptor type, J Protein tyrosine phosphatase, nonreceptor type 2 Protein phosphatase 4, catalytic subunit

0.3

0.2

Ser/Thr

0.2 0.2 0.1 1.2

0.2 0.8 0.5 0.2

Tyr Ser/Thr Tyr Tyr

1.4

0.2

Ser/Thr

Protein phosphatase 1B, magnesium dependent, beta isoform

0.9

0.7

Ser/Thr

NF-kB-Suppressing Phosphatases 1

NM_019411.2

PPP2CA

2

NM_017374.2

PPP2CB

3 4

NM_178726.2 NM_012023.1

PPM1L PPP2R5C

5 6

NM_027982.1 NM_001030985

PP2Cz PPP2R1B

7

NM_026391.2

PPP2R2D

8 9

NM_021529.2 NM_029834.1

G4.1 PPP1R12C

10

NM_011877.1

PTPN21

11

NM_016891.2

PPP2R1A

12 13

NM_030266.1 NM_008960.1

INPP4A PTEN

NF-kB-Activating Phosphatases 14

NM_023200.1

PPP1R7

15 16 17 18

NM_008985.1 NM_023343.1 NM_008982.2 NM_008977.1

PTPRN PP2Cd PTPRJ PTPN2

19

NM_019674.2

PPP4C

Reference Phosphatase 20

NM_011151

PPM1B

Data represent the fold change following RNAi in the NF-kB reporter assay. Astrocytes were treated with medium or 10 ng/ml TNFa. Ser/Thr indicates serine/threonine phosphatase; Tyr, tyrosine phosphatase; Lipid, lipid phosphatase.

confirmed in primary astrocytes. (4) Candidate genes identified by reporter assay were confirmed by use of an independent assay of NF-kB activity. Those genes that scored negative by any of these imposed conditions were excluded from further study. Twenty-five candidate phosphatase genes were identified according to the first criteria (Figures 1B and 1C). These siRNA pairs were further tested by using a mutant NF-kB reporter containing a scrambled NF-kB sequence to exclude effects of the vector backbone. One phosphatase was excluded after RNAi showed >2-fold increases on the mutated NF-kB reporter. Phosphatase expression in astrocytes was confirmed for all but one gene by RT-PCR. Secondary Screen To confirm positive hits from the reporter assay, we investigated the remaining candidates by a chemiluminescent transcription factor binding assay (CTFA), which detects active nuclear NF-kB from astrocytes and its binding ability. Downregulation of 19 phosphatases consistently showed R2-fold changes consistent with the results of the reporter assay. After all screening,

we identified 13 NF-kB-suppressing phosphatases (Figure 1D) and six NF-kB-activating phosphatases (Figure 1E). These genes included 13 components of the serine/threonine phosphatases, four tyrosine phosphatases, and two lipid phosphatases (Table 1 and see Figure S1 in the Supplemental Data available with this article online). Individual siRNA constructs to each of the 19 hits were specific (Figure S2A) and generally reduced target protein or mRNA levels by more than 60% except one PP2Cz siRNA construct that demonstrated only 35% efficiency (Figure S2). Several phosphatases previously known to be involved in NF-kB signaling were identified, including PPP2CA (Yang et al., 2001), PPP4C (Hu et al., 1998), PTPN2 (Ibarra-Sanchez et al., 2001), and PTEN (Mayo et al., 2002), thus underscoring the validity and robustness of our two rounds of screening (Figures 1B and 1C). Moreover, the present study identified eight catalytic and regulatory subunits of PP2A or protein phosphatase 1 (PP1) that modulated NF-kB activity (Table 1). With the exception of PPP1R7, downregulation of all these phosphatase components activated NF-kB transcriptional activity, which is consistent with

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Figure 2. PP2A Catalytic and Structural Components Selectively Form Complexes with IKK and p65 NF-kB (A) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-IKKb antibody. The 50 kDa bands were IgG heavy chain. (B) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-IKKb antibody showing the interaction between endogenous IKK and PP2A. The 25 kDa and 50 kDa bands were IgG light and heavy chains. (C) PPP2CB/PP2R1A core enzyme dephosphorylates Ser181 of IKKb. Astrocytes were transfected with IKKb. Ten minutes after TNFa stimulation, the lysates were immunoprecipitated with anti-IKKb antibody. In a separate transfection, 3T3 cells were transfected with myc-tagged PPP2R1A plus Flag-tagged PPP2CB or other Flag-tagged phosphatases. The phosphatase components were eluted from the Sepharose beads with Flag peptide, and then 2 ng was incubated with immunoprecipitated IKKb for 1 hr at 30 C and immunoblotted with anti-phospho-IKKb antibody. (D) PPP2CB RNAi did not synergize with the constitutively active IKKb SSEE mutant to enhance NF-kB reporter activity. (E) PPP2CB RNAi enhanced IKKb phosphorylation. IKKb-myc was cotransfected with PPP2CB RNAi or control RNAi into astrocytes. After TNFa stimulation, the lysates were harvested and immunoblotted with anti-IKKb antibody and anti-phospho-IKKb antibody.

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the activation of NF-kB and nuclear translocation observed with specific pharmacologic inhibitors of PP1 and PP2A (Sun et al., 1995) (Figures S3A and S3B). We next determined cell specificity by examining the activity of all 19 hits on mouse NIH3T3 fibroblasts (Figure 1F). RNAi to 16 phosphatase genes demonstrated similar effects on both astrocytes and fibroblasts. However, three genes lacked activity on fibroblasts (INPP4A, PTPRN, and PTPRJ). Equally important was the reciprocal observation that PPM1B RNAi enhanced TNFinduced reporter activity in fibroblasts (Figure 1F). This finding is consistent with a previous report that PPM1B associates with the IKK complex in 293T kidney cells, causing dephosphorylation of IKKb and reducing kinase activity (Prajapati et al., 2004). In contrast, PPM1B RNAi was consistently inactive in astrocytes (Table 1). The combined results demonstrate that distinct phosphatase genes can selectively modulate NF-kB responses in various cell types. PP2A Associates with and Dephosphorylates IKK or NF-kB To examine the interactions of the suppressing phosphatases (PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2D, PPP2R5C, G4.1, and PPM1L) or the NF-kBactivating phosphatases (PP2Cd and PPP1R7), the genes were tagged with the myc or Flag epitope. Localization of overexpressed phosphatase components was examined by antiepitope staining. With the exception of PP2Cd, the phosphatases were localized in both the cytoplasm and nucleus of astrocytes. PP2Cd was localized exclusively in nucleus (data not shown). Overexpressed phosphatase genes were next examined for interactions with IKK, NF-kB, and TRAF2 by coimmunoprecipitation. Among the ten phosphatase components tested, only PPP2CB and PPP2R1A pulled down endogenous IKK complexes containing IKKb, IKKg, and IKKa (Figure 2A and data not shown). IKKb was not coprecipitated with the other eight candidates following immunoprecipitation with anti-IKKb antibody (Figure 2A and data not shown). By using antibodies against IKKb or the PP2A catalytic subunit, we detected endogenous IKK complexes containing endogenous PP2A (Figure 2B). Since PPP2CB and PPP2R1A are catalytic and structural subunits of PP2A, respectively, the combined data indicate that a PP2A core enzyme containing PPP2CB and PPP2R1A is associated with the IKK complex. Activation of the IKK complex depends on phosphorylation of its two catalytic subunits, IKKa and IKKb (Delhase et al., 1999). Therefore, we tested whether PPP2CB complexes dephosphorylated IKKb. To examine this, a PP2A core complex containing myc-PPP2R1A was cotransfected along with FlagPPP2CB or other phosphatase components, and the

complexes were eluted from anti-Flag beads with Flag peptide. In vitro studies showed dephosphorylation of IKKb after treatment with purified Flag-PPP2CB plus PPP2R1A (Figure 2C). To further test the specificity of PP2A on IKKb phosphorylation, we cotransfected PPP2CB RNAi constructs with IKKb or the IKKb constitutively active mutant, IKKb S177E/S181E (IKKb SSEE) (Mercurio et al., 1997). We expected that PPP2CB RNAi would synergize with IKKb, but not with the IKKb SSEE mutant in which the phosphorylated serine residues were replaced by glutamic acid. Indeed, as shown in Figure 2D, PPP2CB RNAi synergized with IKKb to increase NF-kB reporter activity, but PPP2CB RNAi failed to synergize with IKKbSSEE. Furthermore, PPP2CB RNAi enhanced basal and TNF-induced IKKb phosphorylation (Figure 2E), suggesting that one mechanism of PPP2CB suppression of NF-kB signaling is through dephosphorylation of the IKK complex by this PP2A phosphatase. Several phosphorylation sites have been identified in NF-kB p65 (Hayden and Ghosh, 2004; Viatour et al., 2005); thus, it was of interest to determine whether phosphatases also controlled NF-kB phosphorylation. The same set of ten phosphatase components was tested by reciprocal coimmunoprecipitation; PPP2CA and PPP2R1B were associated with NF-kB, either by immunoprecipitation with myc antibody or by reverse immunoprecipitation with NF-kB antibody (Figure 2F). PPP2CA showed stronger associations with NF-kB than PPP2R1B (Figure 2F). Endogenous PP2A also formed a complex with endogenous NF-kB p65 (Figure 2G). Phosphorylation of the Ser536 or Ser276 residues in NF-kB p65 is one sign of NF-kB activation (Sakurai et al., 2003; Vermeulen et al., 2003; Zhong et al., 1997). By using an in vitro dephosphorylation assay with phospho-specific antibodies, we found that purified Flag-epitoped PPP2CA/PPP2R1B core enzyme dramatically dephosphorylated the p65 residue Ser536 but failed to dephosphorylate residue Ser276 (Figure 2H). Purified PPP1R7, PP2Cd, and G4.1 had no visible impact on dephosphorylation of either phosphorylation site (Figure 2H). The combined results demonstrate the in vitro specificity of PP2A enzymatic activity on selected residues. Dephosphorylation of TRAF2 by PP2A Holoenzyme Inhibits NF-kB Activity TRAF2 plays an important role in the TNFa-mediated NF-kB signaling pathway. Although TRAF2 is a phosphorylated protein (Chaudhuri et al., 1999; Pomerantz and Baltimore, 1999), the mechanism of TRAF2 phosphorylation and the potential effects of TRAF2 dephosphorylation on NF-kB activity are poorly understood. To address these issues, the association of ten selected

(F) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-NF-kB p65 antibody. The 50 kDa band is IgG heavy chain. (G) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-NF-kB p65 showing the interaction between endogenous NF-kB and PP2A. The 25 kDa and 50 kDa bands were IgG light and heavy chains. (H) PPP2CA /PPP2R1B complexes dephosphorylate Ser536 of p65 NF-kB. Astrocytes were transfected with NF-kB p65. Ten minutes after TNFa stimulation, cells were harvested and immunoprecipitated with anti-NF-kB p65 antibody. In a separate transfection, 3T3 cells were transfected with the Flag-tagged PPP2CA and myc-tagged PPP2R1B or other Flag-tagged phosphatases. These phosphatases were eluted from the Sepharose beads with Flag peptide, and then 2 ng was incubated with immunoprecipitated NF-kB p65 for 1 hr at 30 C and immunoblotted with anti-p65 or two anti-phospho-NF-kB antibodies. The right panel depicts the protein levels of the indicated Flag-tagged phosphatases.

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Figure 3. Dephosphorylation of TRAF2 by PP2A Holoenzyme Inhibits NF-kB Activity (A) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-TRAF2 antibody. (B) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-TRAF2 showing the interaction between endogenous IKK and TRAF2. The 25 kD and 50 kD bands were IgG light and heavy chains. (C) PPP2R5C-myc was cotransfected with Flag-tagged TRAF2 into astrocytes and immunoprecipitated with either anti-Flag or anti-myc antibody. (D) Map of various TRAF2 constructs and their ability to associate with PPP2R5C. (E) Indicated phosphatase genes were cotransfected with TRAF2 into astrocytes. Overexpression of PPP2R5C inhibited TRAF2-induced NF-kB reporter activity. Error bars represent the SD of three independent experiments. (F) After IL-1b stimulation, PPP2R5C RNAi failed to stimulate NF-kB reporter activity in astrocytes.

phosphatases with TRAF2 was tested. As shown in Figure 3A, PPP2CA and PPP2R1A were associated with TRAF2 as noted by immunoprecipitation with anti-myc antibody or by reverse immunoprecipitation with antiTRAF2 antibody. Endogenous PP2A also formed a com-

plex with endogenous TRAF2 (Figure 3B). We also found one PP2A-regulatory subunit, PPP2R5C, associated with the TRAF2 complex (Figure 3C). Further mapping of TRAF2 to evaluate the roles of various functional domains found that both the TRAF-N and TRAF-C domains

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were required for binding of PPP2R5C (Figure 3D). As reported previously, overexpression of TRAF2 induces NF-kB activation, presumably because it induces TRAF2 trimerization, thereby mimicking the effects of ligand stimulation on the TNF receptor (Takeuchi et al., 1996). Co-overexpression of PPP2R5C dramatically inhibited TRAF2-induced NF-kB reporter activity while another PP2A-regulatory subunit (PPP2R2D) and other phosphatases displayed little or no inhibition (Figure 3E). The IL-1 and TNF signal pathways use different TRAF molecules to transduce signals, but the signaling pathways converge further downstream to activate NF-kB. Thus, we tested the effect of PPP2R5C RNAi on IL-1mediated NF-kB reporter activity. As shown in Figure 3F, there was no apparent effect of PPP2R5C RNAi on IL-1-stimulated reporter activity, although PPP2CA, PPP2CB, and PPP2R1B RNAi, which affect the IKK and p65 complexes common to both the TNF and IL-1 signaling pathways, were enhanced (Figure 3F). Weak responses were noted with PPP2R1A: a 2.2-fold increase with IL-1 (Figure 3F) and 1.8-fold with TNF (Table 1). Thus, the functional in vitro data support the physical association of PP2A with TRAF2 and suggest that the PPP2CA/PPP2R1A/PPP2R5C holoenzyme suppresses NF-kB activity by dephosphorylating TRAF2. Previous evidence for TRAF2 phosphorylation was based on a two-dimensional phosphoamino acid separation, which provided little mechanistic insight (Chaudhuri et al., 1999; Pomerantz and Baltimore, 1999). To determine the critical TRAF2 phosphorylation site and corresponding function, we first compared the NF-kB reporter activity of different TRAF2 truncation mutants (Figures S4A and S4B). Consistent with a previous study (Takeuchi et al., 1996), the ring and finger domains were important for TRAF2 activity. To further define the TRAF2 phosphorylation site, 21 conserved serines or threonines were mutated to alanine. Most of these sites were located in the ring and finger domains of TRAF2 (Figure 4A and data not shown). After transfection of these mutants into 293T cells, two mutants, Ser102Ala and Thr117Ala, showed the lowest NF-kB reporter activity (Figure S4C). These two point mutants also showed dramatically reduced NF-kB reporter activity in astrocytes (Figure 4B) and TRAF22/2 MEFs (Figure 4C). To investigate phosphorylation, we noted that the finger domain (residues 99–271) showed two distinct bands by electrophoretic mobility in a 4%–20% SDS-PAGE gel (Figure 4D). The upper band was sensitive to CIP phosphatase treatment (Figure 4D). This suggested that the finger domain of TRAF2 was phosphorylated. Therefore, we generated several finger domain mutants and found that only the Thr117Ala mutation abolished the upper band (Figure 4E). Finally, we generated antibody against a phospho-Thr117 peptide that specifically recognized phosphorylated Thr117 in TRAF2 (Figure 4F and Figure S4D). By using anti-phospho Thr117 antibody, we noted increased TRAF2 Thr117 phosphorylation 15 min after TNFa stimulation (Figure 4G). TNF-induced Thr117 phosphorylation of TRAF2 was inhibited by PPP2R5C overexpression, while neither IKKb Ser181 nor NF-kB p65 Ser536 phosphorylation was affected (Figure 4H). In addition, PPP2R5C RNAi enhanced Thr117 phosphorylation of TRAF2, while control RNAi had no effect on TRAF2 phosphorylation (Figure 4I).

Phosphatases Regulate Chemokine and Cytokine Transcription in Astrocytes Since NF-kB regulates the production of proinflammatory chemokines in astrocytes (Kim et al., 2005; Li et al., 2001; Zhai et al., 2004), the effects of inhibition of the selected ten phosphatases were investigated on chemokine and cytokine transcription. In resting astrocytes, silencing six NF-kB-suppressing phosphatases (PPP2CA, PPP2CB, PPP2R1B, PPP2R2D, PPP2R5C, and PPM1L) enhanced expression of the monocyte chemoattractant MCP-1 and the neutrophil chemoattractant KC, although not always by the 4-fold level used to identify hits in our initial screens (Figure 5A). In contrast, PPP2R1A RNAi increased KC but displayed minimal effects on MCP-1 expression, while G4.1 or PPP1R7 RNAi failed to modulate chemokine levels in resting astrocytes (Figure 5A). In contrast, silencing PP2Cd resulted in reduction of basal MCP-1 and KC mRNA levels (Figure 5A). Silencing of the NF-kB-suppressing phosphatases (PPP2CA, PPP2CB, PPP2R1B, PPP2R2D, and PPP2R5C) also synergized with TNFa for enhanced expression of MCP-1 and KC by >3-fold (Figure 5B). In contrast, G4.1 RNAi selectively enhanced KC expression and PPP1R7 RNAi inhibited TNFa-induced expression of MCP-1 by >70% (Figure 5B). IL-6 expression is tightly regulated, and transcription is dependent on both NF-kB and C/EBP in astrocytes (Schwaninger et al., 2000; Van Wagoner and Benveniste, 1999). Silencing the NF-kB-suppressing phosphatases PPP2CA, PPP2R2D, PPP2R5C, G4.1, and PPM1L increased mRNA levels for IL-6 by >4-fold in resting astrocytes and >3-fold in TNFa-stimulated cells, but IL-6 mRNA was not dramatically enhanced in cells transfected with RNAi to PPP2CB, PPP2R1B, and PPP2R1A (Figures 5A and 5B). In contrast, inhibition of the NFkB-activating phosphatase PPP1R7 resulted in >70% reduction of IL-6 mRNA in resting astrocytes while silencing PP2Cd failed to significantly modulate IL-6 expression in untreated astrocytes (Figure 5A). In summary, silencing of various phosphatase genes resulted in differential patterns of chemokine and/or cytokine regulation; all phosphatase genes examined significantly modulated expression of at least one endogenous chemokine or cytokine.

Discussion Reversible protein phosphorylation is an essential regulatory mechanism in many cellular processes. Cells use this posttranslational modification to alter the activity or localization of key regulatory proteins. Tyrosine and serine/threonine protein phosphatases are highly abundant proteins present in many cellular compartments in mammalian cells. Together with kinases, they set the phosphorylation state of signaling and effector proteins and thereby play a large role in controlling cellular responses. Inappropriate or defective phosphatase or kinase activity leads to aberrant patterns of phosphorylation. Dramatic changes in phosphorylation of many proteins were demonstrated during global ischemia, including enriched phosphatase activity in reactive astrocytes (Hasegawa et al., 2000). To date there has not

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Figure 4. TNF-Induced TRAF2 Thr117 Phosphorylation (A) Amino acid sequence alignment of a portion of the first zinc finger domain of TRAF2. (B) The fold induction of NF-kB reporter activity in astrocytes transfected with TRAF2 and different Ser/Thr to Ala mutants. (C) The fold induction of NF-kB reporter activity in TRAF22/2 MEFs transfected with TRAF2 and different Ser/Thr-to-Ala mutants. Error bars represent the SD of three independent experiments. (D) The zinc finger domain (residues 99–271) showed two bands by electrophoresis in a 4%–20% SDS-PAGE gel. The upper band was sensitive to CIP phosphatase treatment. (E) Thr117-to-Ala mutation abolished the upper band of the first finger domain. (F) Specificity of anti-phospho-TRAF2 (Thr117) antibody. (G) Time course of TNF-induced TRAF2 Thr117 phosphorylation. (H) PPP2R5C inhibited Thr117 phosphorylation. Astrocytes were transfected Flag-TRAF2 with or without PPP2R5C-myc. Cells were treated with or without TNFa for 15 min before harvest and immunoprecipitation with anti-IKKb or anti-NF-kB p65 antibodies and immunoblotted with the indicated antibodies. (I) PPP2R5C RNAi enhanced TRAF2 phosphorylation. TRAF2-Flag was cotransfected with PPP2R5C RNAi or control RNAi into astrocytes. After TNFa stimulation, the lysates were harvested and immunoblotted with anti-Flag antibody and anti-phospho-TRAF2 (Thr117) antibody.

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Figure 5. Phosphatases Regulate Chemokine and Cytokine Expression (A) The relative mRNA levels of MCP-1 (open bars), KC (shaded bars), and IL-6 (solid bars) in RNAi-transfected astrocytes without stimulation. Astrocytes were transfected with phosphatase RNAi vectors and cultured for 72 hr. The cDNAs were analyzed by real-time PCR. All phosphatase mRNA levels were normalized with the housekeeping gene b-glucuronidase. The dashed lines represent a 4-fold increase and 70% reduction, respectively. Error bars represent the SD of three independent experiments. (B) One hour after TNFa stimulation, cells were collected for RNA isolation and subsequent reverse transcription. The relative mRNA levels of MCP-1, KC, and IL-6 in phosphatase RNAi transfected astrocytes after TNFa stimulation. The dashed lines represent a 3-fold increase and 70% reduction, respectively. Error bars represent the SD of three independent experiments. (C) Summary of phosphatase interactions characterized in this report. NF-kB signaling was regulated by dephosphorylation of the TRAF2, IKK, and NF-kB complexes by the indicated PP2A cofactors.

been a systematic examination of phosphatase activity in astrocytes. Here we report a large-scale classification of phosphatases focused on their control of NF-kB-mediated transcriptional activity. Nineteen phosphatases were identified to participate in either up- or downregulation of NF-kB activity in astrocytes. Most of these phosphatases were not previously known to associate with this pathway. The involvement of additional phosphatases cannot be excluded as rigid criteria, and a high threshold of NF-kB activity were used to identify candidate genes.

Stimulus and cell specificity, compensatory or redundant pathways, and the presence of nonfunctional siRNAs may cause additional underestimates of the number of phosphatase genes involved in NF-kB transcriptional activity. At least 13 phosphatases were previously implicated in NF-kB signaling, including PPP2CA (Yang et al., 2001), PPM1B (Prajapati et al., 2004), PPM1L (Li et al., 2003; Takaesu et al., 2003), INPP4A (Franke et al., 1997; Romashkova and Makarov, 1999), PTEN (Mayo et al., 2002), PTPN2 (Ibarra-Sanchez et al., 2001), PPP4C (Hu

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et al., 1998), CDC25B (Zheng et al., 2004), PPP6C (Bouwmeester et al., 2004), PPP2R1A (Zheng et al., 2004), PPP2R1B (Zheng et al., 2004), PPP2R5C (Moreno et al., 2004), and DUSP5 (Zheng et al., 2004). Nine of these genes were also identified by the present analysis, although the mechanisms by which most of these phosphatase genes impact NF-kB signaling are poorly understood. The four genes missed in our screen include DUSP5; however, the murine homolog of DUSP5 has not been identified. Silencing CDC25 phosphatases, which are critical to mitotic entry, markedly inhibited Renilla luciferase activity, suggesting damage to the target cells; therefore, analysis of CDC25B was not pursued. RNAi to PPP6C inhibited basal NF-kB reporter activity but failed to meet the threshold established for our screening. PPM1B (also termed PP2Cb) bound and dephosphorylated IKK in human HeLa and 293 embryonic kidney cells. However, we failed to detect any activity of PPM1B on NF-kB activity in mouse astrocytes (Table 1), even though the RNAi constructs effectively inhibited mRNA levels (Figure S2Z) and modulated NF-kB reporter activity in mouse fibroblasts (Figure 1F). Reciprocally, three phosphatases that regulated NF-kB activity in astrocytes failed to modulate NF-kB reporter activity in fibroblasts. These results suggest potential cell type specificity in the activity of phosphatases on NF-kB signaling, an observation with potential implications for controlling inflammation in various clinical conditions. PP2A enzymes regulate at least three different steps in the NF-kB pathway, including TRAF2, IKK, and NF-kB p65 (Figure 5C). Previous studies showed that the activity of IKK on IkB kinase was associated with PP2A and downregulated by the PP2A catalytic subunit (DiDonato et al., 1997; Fu et al., 2003). We observed selective nonredundant utilization of specific catalytic and structural chains in the core enzyme complexes, i.e., PPP2CB/PPP2R1A were selectively coupled to the IKK complex while PPP2CA/PPP2R1B were physically and functionally associated with the p65 NF-kB complex (Figure 2). Although the PP2A complex was shown to bind and dephosphorylate the p65 chain of NF-kB (Yang et al., 2001), there was no description of the composition of the PP2A enzyme. The present report functionally extends these observations by identifying PPP2CA and PPP2R1B as the NF-kB interactive chains (Figure 2) and demonstrates the selective dephosphorylation of the Ser536 residue in the NF-kB p65 subunit. Our data suggest the potential of multiple corresponding site-specific phosphatases for NF-kB p65. In addition, we identified a PP2A holoenzyme associated with TRAF2 (Figure 3). Analysis of this interaction demonstrated that Thr117 in the first TRAF2 zinc finger domain is a phosphorylation site and phosphorylation of Thr117 is required for TRAF2-mediated NF-kB activity (Figure 4). The present data also demonstrate ligandinduced phosphorylation of TRAF2 and suggest that TRAF2 may be the target of the PP2A holoenzyme (Figure 4). Future experiments will address the mechanisms involved in TRAF2 phosphorylation. The PP2A chains combine in different combinations to form core enzymes and holoenzymes. In mice, the PPP2CA and PPP2CB catalytic chains are 97% identical, and the structural chains are 86% identical. However, PPP2CA null mutant mice were embryonic lethal,

demonstrating that PPP2CA is an essential nonredundant gene (Gotz et al., 1998). In the present study, coimmunoprecipitation showed that nonredundant PP2A catalytic and structural chains were preferentially associated with their substrate. This suggests that selective combinations of nonredundant PP2A catalytic and structural chains may be critical for substrate targeting. Several phosphatases regulated basal NF-kB activity, suggesting that NF-kB activity is tightly regulated and may be required for cellular homeostasis. Indeed, phosphorylation of p65 and its shuttling in and out of the nucleus have been observed in several cell types including astrocytes (Zhai et al., 2004). Basal NF-kB activity was reported to be critical for protecting cells from apoptosis (Bureau et al., 2002). Constitutive NF-kB activity has also been detected in glioblastomas and other tumors. The molecular mechanisms responsible for altered regulation of the NF-kB pathway in cancer cells remain largely unknown, but some phosphatase genes (e.g., PTPRJ and PPP2CB) identified in this report sensitize or promote cell death (MacKeigan et al., 2005) and therefore hold potential roles as tumor suppressors. Astrocytes are implicated in the pathophysiology of neurodegenerative and inflammatory diseases including Alzheimer’s disease and multiple sclerosis (Miller, 2005). These diseases are characterized by scarring lesions containing reactive hypertrophic astrocytes. These reactive astrocytes are a major source of chemokines that orchestrate migration and activation of leukocytes and microglial cells into neuronal lesions. The knowledge that phosphatases identified in this report can selectively regulate chemokine and cytokine expression (Figure 5) offers new therapeutic targets with the potential of regulating inflammatory diseases. Experimental Procedures Mice and Astrocyte Isolation BALB/cByJ mice (Jackson Laboratory, Bar Harbor, Maine) were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School. Astrocytes were prepared from neonatal (<24 hr) mice, as previously described (Luo et al., 2002). The purity of the primary astrocyte cultures was >95%, as determined by indirect immunofluorescence with anti-GFAP antibody (Dako, Carpinteria, California) (Figure S3B). NF-kB Reporter Gene Design The RNAi screen was based on the pLuc-MCS reporter (Stratagene, La Jolla, California), which consists of a basic TATA element driving expression of a cDNA encoding the firefly luciferase gene. To optimize this assay in a 96-well plate format, we designed six pNF-kBLuc-like reporters containing 1, 2, 6, 12, 18, or 24 NF-kB p65 binding sites, respectively. Although these reporters showed different basal activities, the reporter containing six binding sites exhibited the highest signal to noise ratio and was used in reporter screens. siRNA Vector Design We modified the dual siRNA retrovirus-based expression vector named pBabe-Dual (pBabe-puro with dual RNA polymerase III promoter). It contains two opposing RNA polymerase III promoters to drive expression of both strands of a template DNA cloned between the promoters. Both the H1 and U6 promoters were modified to contain a five thymidine Pol III termination sequence at the 25 to 21 position and two BbsI sites in the insertion (Figure 1A). The target sequence for any mRNA can be cloned into pBabe-Dual, and the DNA will be transcribed from both strands to form a doublestranded RNA with two 30 uridine overhangs. The efficiency of inhibition of this siRNA vector was determined by RNAi experiments

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in which firefly luciferase and GFP were inhibited. By using this system, RNAi-mediated knockdown of a positive regulator (NF-kB p65) suppressed TNFa-stimulated reporter activity while RNAi knockdown of a negative regulator (IkBa) activated the reporter in the absence of stimulus or synergistically activated the reporter when induced by TNFa (data not shown). siRNA Library Construction and Screen All 250 mouse phosphatase genes and putative phosphatase genes were chosen from the public UniGene library. siRNA target sequences in the gene were chosen by using the siRNA design program from the Whitehead Institute web page, http://jura.wi.mit. edu/siRNAext/home.php. Two siRNA target sequences were designed for each phosphatase gene by using the following criteria. (1) The selected siRNA sequences for a given gene should not have more than 85% similarity to any other gene in mouse UniGene database by using the Blast program. (2) Sequences were selected with 40%–65% GC content. (3) No sequence containing four or more sequential bases of the same nucleotide was allowed. (4) No thermodynamically stable secondary structure (<0 Kcal/mol) was allowed. (5) A 50 terminus on the antisense strand that is more AT rich than the 30 terminus. The siRNA sequences are available on request. A p6XNF-kB-Luc reporter plasmid was selected to screen for regulators of NF-kB transcriptional activation. A Renilla luciferase plasmid and two siRNA constructs for each gene target were combined and cotransfected into mouse astrocyte cells for screening. Forty-eight hours after transfection, the cells were starved overnight and then stimulated for 6–8 hr with 10 ng/ml TNFa, and luciferase activity was subsequently measured. To screen for regulators of basal NF-kB transcriptional activity, cells were not stimulated and luciferase activity was measured 72 hr after transfection. Data Analysis Normalized values (N), where N = (firefly luciferase value)/(Renilla luciferase value), were calculated as described by DasGupta et al. (2005). We chose this log transformation analysis because the data fit in a linear progression for both increases and decreases with respect to the plate average. Genes scoring >2 SD from the average (log[N]) were considered potential hits. Cells and Reagents NIH3T3 cells were purchased from American Type Culture Collection (ATCC) (Manassas, Virginia). TRAF22/2 MEFs were kindly provided by Dr. Tak Mak (University of Toronto). Recombinant mouse and human TNFa were purchased from R&D Systems (Minneapolis, Minnesota). Okadaic acid and Calyculin A were obtained from Calbiochem (La Jolla, California). DAPI was purchased from Sigma Chemical Co. (St. Louis, Missouri). Anti-phospho-TRAF2 (Thr117) antibody was generated in rabbits (Convance, Denver, Pennsylvania) by using synthetic phosphopeptide CTWKGT*LKEYE (T*: phospho-T) conjugated to keyhole limpet hemocyanin as immunogen. Immune serum was passed through a phosphopeptide affinity column and washed with 0.1 M Tris (pH 8.0). Bound antibodies were eluted with 0.2 M glycine (pH 2.5) and neutralized with 1 M Tris (pH 8.0). Antibodies directed to IkBa, phospho-IkBa (Ser32/Ser36), IKKa, IKKb, IKKg, phospho-IKKa (Ser180)/b (Ser181), TRAF2, phospho-p65 (Ser276), and phospho-p65 (Ser536) were bought from Cell Signaling (Beverly, Massachusetts). Antibodies specific for myc or p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Antibodies specific for Flag were purchased from Sigma Chemical Co. All phosphatase cDNAs used for overexpression were ordered from ATCC and tagged with the myc epitope in the pcDNA3.1myc-His vector. IKKb-Flag was kindly provided by Dr. R.B. Gaynor (Lilly Research Laboratories). IKKb SSEE (Mercurio et al., 1997) was purchased from Addgene (Cambridge, Massachusetts). Real-Time RT-PCR mRNA was quantified by using SYBR Green-based real-time PCR. Total RNA was prepared by using TRIzol Reagent (Invitrogen, Carlsbad, California). Two micrograms of RNA was transcribed into cDNA by using 200 U Superscript II (Invitrogen Life Technologies). For one real-time reaction, a 20 ml SYBR Green PCR Reaction Mix (Roche Applied Science) was supplemented with 1/40 of the synthesized

cDNA plus an appropriate oligonucleotide primer pair and run on the LightCycler II (Roche). Reverse transcriptase controls were done in parallel without adding enzyme. The comparative Ct method was used to determine relative mRNA expression of examined genes as normalized by the b-glucuronidase housekeeping gene. Cell Transfection and Luciferase Activity Assay Astrocytes were transiently transfected with Lipofectamine 2000 (Invitrogen). Forty-eight hours later, the cells were starved overnight and then stimulated for the indicated time. Luciferase activity was determined as recommended by the manufacturer (Promega, Madison, Wisconsin). Values are expressed as mean 6 SD of three experiments. Luciferase assays were performed by using the Dual Luciferase reporter system (Promega). Relative luciferase units (RLU) were measured and normalized against Renilla luciferase activity 72 hr after transfection. Immunoblotting, Immunoprecipitation, and Immunocytochemistry Cells were harvested and analyzed by western blot. Protein concentration was determined by BCA protein assay kit (Pierce, Rockford, Illinois). Samples (10 mg) were loaded in each lane. Blots were probed with the indicated antibody, and immune complexes were detected by enhanced chemiluminescence (ECL) Plus (Amersham Pharmacia Biotech, Piscataway, New Jersey). For the NF-kB nuclear translocation assay, astrocytes were grown on glass slide chambers for 2 days after transfection. After TNFa treatment, cells were fixed in 4% paraformaldehyde and permeablized in phosphate-buffered saline (PBS) containing 0.1% Triton X-100. After blocking with 5% normal goat serum in PBS, cells were incubated with anti-p65 antibody, followed by incubation with Cy2- or Cy3-conjugated goat anti-rabbit IgG (Chemicon). Nuclei were stained with 100 ng/ml DAPI in PBS for 5 min. Immunoprecipitation kits (protein G) were purchased from Roche, and immunoprecipitation was performed according to the manufacturer’s protocol. Chemiluminescent Transcription Factor Assays EZ-Detect Transcription Factor Kit for NF-kB p65 was purchased from Pierce Biotechnology (Rockford, Illinois). Assays were performed according to the manufacturer’s protocol. Supplemental Data Supplemental Data include four figures and can be found with this article online at http://www.molecule.org/cgi/content/full/24/4/ 497/DC1/. Acknowledgments This work was supported by NIH grant 1 RO1 NS42900 and NMSS grant RG2989B3/1. We thank Dr. T. Mak (University of Toronto) and Dr. R.B. Gaynor (Lilly Research Laboratories) for their generous gift of reagents. Received: May 30, 2006 Revised: August 11, 2006 Accepted: October 11, 2006 Published: November 16, 2006 References Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P.O., Bergamini, G., Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., et al. (2004). A physical and functional map of the human TNF-alpha/ NF-kappa B signal transduction pathway. Nat. Cell Biol. 6, 97–105. Bureau, F., Vanderplasschen, A., Jaspar, F., Minner, F., Pastoret, P.P., Merville, M.P., Bours, V., and Lekeux, P. (2002). Constitutive nuclear factor-kappaB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteins. Blood 99, 3683–3691. Bush, T.G., Puvanachandra, N., Horner, C.H., Polito, A., Ostenfeld, T., Svendsen, C.N., Mucke, L., Johnson, M.H., and Sofroniew, M.V. (1999). Leukocyte infiltration, neuronal degeneration, and neurite

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