Cytoplasmic linker protein 170 (CLIP170) is a CAP-Gly domain–containing protein that is associated with the plus end of growing microtubules and implicated in various cellular processes, including the regulation of microtubule dynamics, cell migration, and intracellular transport. Our studies revealed a previously unrecognized property and role of CLIP170. We identified CLIP170 as one of the interacting partners of Brucella effector protein TcpB that negatively regulates TLR2 and TLR4 signaling. In this study, we demonstrate that CLIP170 interacts with the TLR2 and TLR4 adaptor protein TIRAP. Furthermore, our studies revealed that CLIP170 induces ubiquitination and subsequent degradation of TIRAP to negatively regulate TLR4-mediated proinflammatory responses. Overexpression of CLIP170 in mouse macrophages suppressed the LPS-induced expression of IL-6 and TNF-α whereas silencing of endogenous CLIP170 potentiated the levels of proinflammatory cytokines. In vivo silencing of CLIP170 in C57BL/6 mice by CLIP170-specific small interfering RNA enhanced LPS-induced IL-6 and TNF-α expression. Furthermore, we found that LPS modulates the expression of CLIP170 in mouse macrophages. Overall, our experimental data suggest that CLIP170 serves as an intrinsic negative regulator of TLR4 signaling that targets TIRAP.

Toll-like receptors are essential components of innate immune response, which is at the first level of host defense against invading microorganisms. TLRs carry numerous leucine-rich repeats containing an extracellular domain that binds specific conserved pathogen-associated molecular pattern and an intracellular Toll/IL-1 receptor (TIR) domain that interacts with specific adaptor proteins such as MyD88, TIR domain–containing adaptor protein (TIRAP), TIR domain–containing adaptor inducing IFN-β, TRAM, and SARM to initiate the signaling (13). TIRAP is essential for TLR2- and TLR4-mediated signaling where it recruits MyD88 to the TIR domain of plasma membrane localized TLR2 and TLR4 (4). Activation of TLR signaling by pathogen-associated molecular patterns leads to the activation of transcription factors, including NF-κB, that in turn triggers expression of many proinflammatory cytokine genes (5). Subsequent secretion of proinflammatory cytokines activates innate immune cells that leads to various antimicrobial responses and promotes adaptive immunity.

TLR activities are tightly regulated to maintain cellular homeostasis following activation by microbial components. Ubiquitination and deubiquitination play essential roles in the regulation of TLR signaling pathways (6, 7). Ubiquitination comprises covalent attachment of ubiquitin to the lysine residues of target proteins, which involves activation of ubiquitin residues by ubiquitin-activating enzyme (E1) followed by transfer of activated ubiquitin to the catalytic cysteine of the ubiquitin-conjugating enzyme (E2) (8). Subsequently, the ubiquitin-conjugating enzyme transfers the ubiquitin moiety to the lysine residues of target proteins, which are catalyzed by ubiquitin ligases (E3). Ubiquitination can be monoubiquitination where a single ubiquitin moiety is attached to the substrate, or polyubiquitination where polymerized ubiquitin chains are linked through the lysine residues of ubiquitin to the target protein. Ubiquitination and proteosomal degradation of TLRs or adaptor proteins have been implicated in negative regulation of TLR signaling to restore cellular homeostasis. Triad3A is an E3 ubiquitin ligase that negatively regulates TLR4 and TLR9 signaling by promoting their K48-linked ubiquitination and proteolytic degradation (9). Triad3A was also reported to induce proteolytic degradation of TIRAP (10). Suppressor of cytokine signaling-1 (SOCS-1) inhibits TLR2 and TLR4 receptor signaling by inducing polyubiquitination and degradation of the adaptor protein TIRAP (11).

Brucella melitensis, an infectious intracellular bacterial pathogen, encodes the TIR domain–containing protein TcpB that suppresses TLR2 and TLR4 signaling to subvert host innate immune responses (1216). TcpB targets the TLR2 and TLR4 adaptor protein TIRAP and induces ubiquitination and subsequent degradation of TIRAP (12, 17). However, the mechanism of TcpB-mediated ubiquitination of TIRAP remains obscure. We performed a high-throughput yeast two-hybrid screening that identified the microtubule plus end tracking protein, cytoplasmic linker protein 170 (CLIP70), as the interacting partner of TcpB. CLIP170 is a microtubule-associated protein that specifically binds to plus ends of growing microtubules (18). It is characterized by two conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domains present at the N terminus that function as the microtubule binding module (19). CLIP170 harbors two tandem repeated zinc knuckle motifs in the C terminus that have been implicated in the interaction with endocytic vesicles and other microtubule plus-end-tracking proteins (2022). CLIP170 is a multifunctional protein that plays essential roles in regulating microtubule dynamics, dynein localization to the microtubule tips, microtubule interaction with the cell cortex, and linking of endosomes to microtubules (18, 2328). In this study, we report that CLIP170 enhances the ubiquitination of TIRAP, which promotes the proteasome-mediated degradation of TIRAP. Furthermore, we demonstrate that CLIP170 serves as a negative regulator of TLR4 signaling, and CLIP170 expression is modulated by the TLR4 ligand LPS.

Human embryonic kidney (HEK) 293T (American Type Culture Collection) or HEK293FT cells (Thermo Fisher Scientific) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 1× penicillin-streptomycin solution (Life Technologies), and 100 μg/ml normocin (InvivoGen). RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS and 1× penicillin-streptomycin solution (Life Technologies) was used to culture J774 mouse macrophages (American Type Culture Collection) and mouse embryonic fibroblast (MEF) cells (American Type Culture Collection). Cells were grown in a 37°C humidified atmosphere of 5% CO2. All transfections were performed using Lipofectamine 2000 (Invitrogen) or JetPEI macrophage (Polyplus Transfections) according to the manufacturers’ instructions.

To analyze interaction between TIRAP and CLIP170, HEK293T cells (1 × 106) were cotransfected with FLAG-TIRAP (gifted by Dr. D. Golenbock) and hemagglutinin (HA)-CLIP170 or empty vector using Lipofectamine 2000 reagent in six-well plates. Forty-eight hours after transfections, cells were lysed and clarified by centrifugation. The cell lysates were precleared with protein G plus agarose beads followed by addition of 5 μg of anti-FLAG Ab (Sigma-Aldrich) and incubation overnight at 4°C on a rotator. Next, protein G plus agarose beads were added into the samples and incubated further for 3 h at 4°C on a rotator. Subsequently, agarose beads were washed three times with TNT buffer (20 mM Tris [pH 8], 150 mM NaCl, 1% Triton X-100) and resuspended in 30 μl of SDS sample buffer (Bio-Rad Laboratories) and boiled for 10 min followed by SDS-PAGE and immunoblotting. The membrane was probed with HRP-conjugated anti-HA Ab (Sigma-Aldrich) in 5% milk in TBST overnight at 4°C. Subsequently, the membrane was washed three times with TBST for 5 min each and incubated with SuperSignal West Pico chemiluminescent substrate (Pierce) for 5 min followed by acquiring the luminescent signal using a chemiluminescence documentation system (Syngene). After that, the membrane was treated with Restore Western blot stripping buffer (Pierce) and reprobed with anti-FLAG Ab to detect FLAG-TIRAP. The whole-cell lysates were also subjected to immunoblotting and probed with HRP-conjugated anti-HA and anti-FLAG Abs to detect HA-CLIP170 and FLAG-TIRAP, respectively.

HEK293T cells (0.5 × 106) were seeded into 12-well plates and cotransfected with 300 ng of FLAG-tagged TIRAP/MyD88 (gifted by Dr. D. Golenbock)/human TLR2 (gift from R. Medzhitov, plasmid no. 13082; Addgene)/TLR4/murine TLR9 (gift from R. Medzhitov, plasmid no. 13091; Addgene) plasmids and increasing concentrations (300, 600, 900 ng, and 1.2 μg) of HA-CLIP170 plasmid. Twenty-four hours after the transfections, cells were lysed in RIPA buffer and the protein concentration was estimated using Bradford reagent. Equal amounts of protein samples were loaded on 4–20% Tris-glycine SDS-PAGE gel and subjected to immunoblotting. The membrane was probed with HRP-conjugated anti-FLAG Ab (no. A8592; Sigma-Aldrich) to detect FLAG-tagged proteins and HRP-conjugated anti-HA Ab (no. H6533; Sigma-Aldrich) for detecting HA-CLIP170. Actin was detected using monoclonal anti–β-actin peroxidase-conjugated Ab (no. A3854; Sigma-Aldrich).

To analyze the degradation of endogenous TIRAP or MyD88, RAW264 cells (0.5 × 106) were seeded into 12-well plates and transfected with 3 μg of HA-CLIP170 plasmid or empty vector using FuGENE HD transfection reagent (Promega) according to the manufacturer’s protocols. Twenty-four hours after transfections, cells were lysed in RIPA buffer and subjected to immunoblotting. Endogenous TIRAP was detected using anti-TIRAP Abs (no. 13077S, Cell Signaling Technology; no. sc-166149, Santa Cruz Biotechnology; or no. ab17218, Abcam). Anti-rabbit IgG, HRP-linked Ab (no. 7074; Cell Signaling Technology) was used as the secondary Ab for the blots probed with anti-TIRAP Ab from Cell Signaling Technology and Abcam. Anti-mouse IgG, HRP-linked Ab (no. 7076; Cell Signaling Technology) was used as the secondary Ab for the blot probed with anti-TIRAP Ab from Santa Cruz Biotechnology. Endogenous MyD88 was detected using anti-MyD88 Ab (no. sc-74532; Santa Cruz Biotechnology).

Total RNA was isolated from HEK293T cells transfected with FLAG-TIRAP plus HA-CLIP170 or FLAG-MyD88 plus HA-CLIP170 plasmids followed by cDNA synthesis using a PrimeScript first-strand cDNA synthesis kit (Takara Bio). Primers used for amplification are CLIP170 (forward, 5′-GTGGCGTGGAGTTAGATGAG-3′; reverse, 5′-TTTGGCTGGTGTAGTGGAAG-3′), TIRAP (forward, 5′-CTCCTACTTGGAAGGCAGCAC-3′; reverse, 5′-ACGAAAGCCACCATCAGGG-3′), MyD88 (forward, 5′-TGCTGGAGCTGGGACCCAGCATTGAGGA-3′; reverse, 5′-TCAGACACACACACAACTTCAGTCGATA-3′), and GAPDH (forward, 5′-AATCCCATCACCATCTTCCA-3′; reverse, 5′-TGGACTCCACGACGTACTCA-3′). Relative gene expression of CLIP170, TIRAP, and MyD88 was analyzed by the comparative 2−ΔΔCt method using ABI 7500 software (Applied Biosystems). Data were normalized with an endogenous control, GAPDH.

HEK293FT cells (1 × 106 in six-well plates) were cotransfected with 1.5 μg of MYC-CLIP170, 1.5 μg of FLAG-TIRAP or FLAG-MyD88, and 1 μg of wild-type or mutant versions of HA-ubiquitin (gifted by Dr. S. Miyamoto) plasmids in various combinations, identified in the results. Total DNA concentration was maintained at 4 μg using the empty vector. Twenty hours after the transfection, the cells were treated with proteasome inhibitor MG132 (Sigma-Aldrich) at a concentration of 20 μM for 4 h. Cells were then washed once with PBS and lysed in 300 μl of lysis buffer containing 20 mM Tris-HCI (pH 7.4) and 1% SDS (17). Cell lysates were transferred into Eppendorf tubes and boiled for 10 min. Lysates were then clarified by centrifugation at 13,000 rpm for 15 min and diluted with buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2% Triton X-100, and 0.5% Nonidet P-40 (17). Five micrograms of anti-FLAG Ab was added into the lysates and incubated overnight at 4°C on a rotator. Immunoprecipitation and Western analysis were performed with samples as described before. The membrane was probed with HRP-conjugated anti-HA Ab to detect HA-ubiquitin.

MEF cells (1 × 106) were seeded into 30-mm glass-bottom petri plates (Eppendorf) and allowed to adhere overnight. Cells were then transfected with 3 μg of MYC-CLIP170 or HA-TcpB plasmid using Lipofectamine 3000 reagent. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 min, followed by three washes for 5 min each with PBS and incubated with 50 mM NH4Cl for 10 min. Cells were then permeabilized with 0.1% Triton X-100 for 5 min. Subsequently, cells were washed three times with PBS and incubated with FITC-conjugated anti-MYC Ab (no. F2047; Sigma-Aldrich) to stain MYC-tagged proteins, FITC-conjugated anti-HA (no. H7411; Sigma-Aldrich) to stain HA-TcpB, and Cy3-labeled anti–β-tubulin Ab (no. C4585; Sigma-Aldrich) to stain microtubules for 1 h. Cells were washed and mounted in ProLong Gold antifading reagent (Molecular Probes). Cells were analyzed using a confocal microscope (Leica SP8). Experiments were repeated three times and 15–20 cells were analyzed per experiment.

For TLR4 activation assays, 12 h prior to the transfections, HEK293T cells expressing human TLR4, CD14, and MD2 (293/hTLR4A-MD2-CD14; InvivoGen) were seeded into 12-well plates at a density of 0.5 × 106 per well. Cells were then cotransfected with various amounts of HA-CLIP170 (50, 100, and 250 ng), pNF-κB–Luc (100 ng; Stratagene), and pRL-TK (50 ng; Promega) plasmids. The total amount of DNA was made constant by adding empty vector (pCMV-HA). Twenty-four hours posttransfection, cells were challenged with 300 ng of LPS for 12 h. Cells were then lysed and luciferase activity was assayed using the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s protocols. Firefly luciferase activity was normalized to the Renilla luciferase activity and the reporter assays were expressed as mean fold induction over uninduced controls. For TLR9 activation assays, HEK293T cells were cotransfected with murine TLR9 (100 ng) expression plasmid and other reporter plasmids mentioned above followed by induction with 1 μg of oligodeoxynucleotides (ODNs) and measurement of luciferase activity. Assays were performed in triplicate, and the experiments were repeated thrice.

To overexpress CLIP170 in mouse macrophages, a full-length CLIP170 coding DNA sequence segment was ligated to the lentivirus expression plasmid, pMSCV-PIG (a gift from D. Bartel, plasmid no. 21654; Addgene). To prepare lentiviral particles harboring pMSCV-PIG-CLIP170, HEK293FT cells (1 × 106 in six-well plates) were cotransfected with packaging vector psPAX2 (a gift from D. Trono, plasmid no. 12260; Addgene) and the envelope vector and pMD2.G (a gift from D. Trono, plasmid no. 12259; Addgene) and pMSCV-PIG-CLIP170 or empty vector. Seventy-two hours after transfection, culture supernatant that contains lentiviral particles was collected and clarified by centrifugation and passed through a 0.45-μm syringe filter (Millipore). The lentiviral titer in the clarified cell supernatant was assessed using Lenti-X GoStix (Clontech) followed by transduction of J774 cells (1 × 106) with lentiviral particles. Forty-eight hours after transduction, cells were lysed in RIPA buffer and analyzed by immunoblotting to confirm the overexpression of CLIP170 using anti-CLIP170 Ab (no. sc-28325; Santa Cruz Biotechnology). To analyze the overexpression of CLIP170 by quantitative PCR (qPCR), total RNA was extracted from lentivirus-transduced cells followed by cDNA synthesis and qPCR analysis. Primers used for CLIP170 amplification are as follows: CLIP, forward, 5′-CCTCAAGATGAAGGTGGAGATG-3′, reverse, 5′-AGGTCAAAGCAGTCACAGATG-3′. CLIP170 qPCR primers were derived from a region that spans the exon–intron boundary of pre-mRNA of CLIP170 transcript.

To study effect of CLIP170 on cytokine secretion by macrophages, J774 cells were transduced with CLIP170 or empty vector for 48 h, followed by treatment with LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), ODN (1 μg/ml), or TNF-α (20 ng/ml). Subsequently, culture supernatants and cells were collected at 0, 1, and 2 h after treatment. Total RNA was extracted from the LPS-challenged cells followed by cDNA synthesis and qPCR analysis. Primers for qPCR analysis were: TNF-α, forward, 5′-GACGTGGAAGTGGCAGAAGAG-3′, reverse, 5′-TGCCACAAGCAGGAATGAGA-3′; IL-6, forward, 5′-TCCAGTTGCCTTCTTGGGAC-3′, reverse, 5′-GTACTCCAGAAGACCAGAGG-3′; IFN-β, forward, 5′-CTGGCTTCCATCATGAACAA-3′, reverse, 5′-CATTTCCGAATGTTCGTCCT-3′; and murine GAPDH, forward, 5′-AACGACCCCTTCATTGAC-3′, reverse, 5′-TCCACGACATACTCAGCAC-3′. IL-6, TNF-α, IFN-β, and CLIP170 expression data were normalized to the expression levels of GAPDH. Quantification of IL-6 and TNF-α in the cell culture supernatant was performed by ELISA using a Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocols. All assays were performed at least three times.

To silence the endogenous CLIP170 in J774 cells, we synthesized CLIP170-specific short hairpin RNA (shRNA), derived from CLIP170 small interfering RNA (siRNA) reported previously (23). The nontargeting control shRNA sequence used was 5′-CCGGTTCTCCGAACGTGTCACGTTTCTGCAGAAACGTGACACGTTCGGAGAATTTTTG-3′. The shRNA oligonucleotides were annealed and cloned into pLKO-1 vector (a gift from D. Root, plasmid no. 10878; Addgene). Lentiviral particles harboring the CLIP170 shRNA or control shRNA were prepared as described above. J774 cells were transduced with lentivirus, and silencing of endogenous CLIP170 was assessed by immunoblotting using anti-CLIP170 Ab and qPCR. To analyze the levels of IL-6 and TNF-α in CLIP170-silenced cells, J774 cells were transduced with lentivirus harboring CLIP170-specific shRNA or control shRNA. Forty-eight hours after the transduction, cells were challenged with various ligands followed by quantification of IL-6, TNF-α, and IFN-β in the culture supernatants and cells by ELISA and qPCR, respectively, as described above.

Mouse experiments were performed after obtaining Institutional Animal Ethics Committee approvals. The in vivo siRNA targeting the CLIP170 (5′-GGAGAAGCAGCAGCACAUUTT-3′) and scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were synthesized from Ambion. In vivo siRNA was complexed with the in vivo siRNA delivery reagent, Invivofectamine 2.0 (Invitrogen), according to the manufacturer’s protocols. One hundred microliters of siRNA complex that contained 50 μg of siRNA was injected into 6-wk-old female C57BL/6 mice through the tail vein. To examine the silencing of endogenous CLIP170 in the liver, 48 h after the siRNA delivery, Kupffer cells were harvested from the mice employing a gradient centrifugation protocol as described by Li et al. (29). Adhered Kupffer cells were lysed and subjected to immunoblotting using anti-CLIP170 Ab. Spleens were also harvested and dispersed in RPMI 1640 in 30-mm culture dishes. Spleen homogenate was then filtered through a cell strainer and seeded into six-well plates. Splenocytes were allowed to adhere for 2 h followed by cell lysis and immunoblotting.

To analyze levels of IL-6 and TNF-α in splenocytes, cells were prepared as described before. Cells were then challenged with LPS (100 ng/ml) followed by quantification of IL-6 and TNF-α levels in the culture supernatants and mRNA in cells by ELISA and qPCR, respectively. To analyze the levels of proinflammatory cytokines in the liver of siRNA-treated mice, mice were injected i.p. with sublethal dose of LPS (25 mg/kg) after 72 h of siRNA delivery. Two hours after the LPS injection, mice were sacrificed followed by collection of liver. Total RNA was isolated from the liver and cDNA was synthesized. The levels of IL-6 and TNF-α mRNA were measured in the liver by qPCR analysis.

J774 cells (0.5 × 106) were seeded into 12-well plates and allowed to adhere overnight. Cells were then challenged with LPS (100 ng/ml) for various times (0, 15, 30, 60, and 120 min) followed by lysis in RIPA buffer for immunoblotting or isolation of total RNA for qPCR analysis. For immunoblot analysis, membranes were probed with anti-CLIP170 Ab followed by anti–β-actin Ab where β-actin served as the loading control. For qPCR analysis, cDNA was prepared from total RNA followed by quantification of endogenous levels of CLIP170.

We analyzed the interaction between CLIP170 and the TLR2/4 adaptor protein TIRAP using coimmunoprecipitation assays. HEK293T cells were cotransfected with FLAG-TIRAP and HA-CLIP170 followed by immunoprecipitation with anti-FLAG Ab. HA-CLIP170 could be coimmunoprecipitated with FLAG-TIRAP, indicating potential interaction between CLIP170 and TIRAP (Fig. 1A). SOCS-1 promoted polyubiquitination and subsequent degradation of TIRAP to negatively regulate TLR4 signaling (11). The Brucella effector protein TcpB that interacted with CLIP170 also induced enhanced ubiquitination and degradation of TIRAP (17). Therefore, we examined whether CLIP170 promotes degradation of TIRAP. We cotransfected HEK293T cells with a constant amount of FLAG-TIRAP and increasing concentrations of HA-CLIP170 followed by immunoblot analysis using anti-FLAG Ab to detect the FLAG-TIRAP. We observed enhanced degradation of FLAG-TIRAP with increasing concentrations of HA-CLIP170 (Fig. 1B). To rule out the possibility that CLIP170 may affect the expression of TIRAP, we analyzed the TIRAP mRNA levels in the presence or absence of CLIP170. TIRAP mRNA levels were not altered by overexpression of CLIP170 (Supplemental Fig. 1).

FIGURE 1.

CLIP170 induces ubiquitination and degradation of TIRAP. (A) CLIP170 interacts with TIRAP. HEK293T cells were cotransfected with equal amounts of HA-CLIP170 and FLAG-TIRAP plasmids. Twenty-four hours posttransfection, cells were lysed and FLAG-TIRAP was immunoprecipitated using anti-FLAG Ab followed by immunoblotting. The blot was probed with anti-HA Ab to detect the coimmunoprecipitated HA-CLIP170 followed by detection of FLAG-TIRAP by anti-FLAG Ab. The whole-cell lysates were also subjected to immunoblotting followed by immunodetection of HA-CLIP170 and FLAG-TIRAP. (B) CLIP170 promotes degradation of TIRAP. HEK293T cells were cotransfected with FLAG-TIRAP and increasing concentrations of HA-CLIP170 plasmids. Twenty-four hours posttransfection, cells were lysed and subjected to immunoblotting. The blot was probed with anti-FLAG and anti-HA Abs to detect FLAG-TIRAP and HA-CLIP170, respectively. Actin served as the loading control. The right panel of the immunoblot shows the densitometry analysis of FLAG-TIRAP bands normalized to actin. (C) CLIP170 induces ubiquitination of TIRAP. HEK293T cells were cotransfected with various combinations of FLAG-TIRAP, MYC-CLIP170, and HA-ubiquitin as indicated. Twenty-four hours posttransfection, cells were treated with 20 μM MG132 for 4 h as indicated in the figure. Cells were then lysed and FLAG-TIRAP was immunoprecipitated followed by immunoblotting. The blot was probed with anti-HA Ab to detect the HA-ubiquitin–conjugated FLAG-TIRAP. CLIP170 enhanced the ubiquitination of CLIP170 in MG132-treated or untreated cells. Similar concentrations of FLAG-TIRAP and MYC-CLIP170 resulted in accumulation of ubiquitinated FLAG-TIRAP in the immunoprecipitated samples. (D) CLIP170 did not induce degradation of MyD88. HEK293T cells were cotransfected with 300 ng of FLAG-MyD88 and increasing concentrations of HA-CLIP170. Twenty-four hours posttransfection, cells were lysed and subjected to immunoblotting. The blot was probed with anti-FLAG and anti-HA Abs to detect FLAG-MyD88 and HA-CLIP170, respectively. Degradation of MyD88 was not observed with increasing concentrations of HA-CLIP170. The right panel of the immunoblot shows the densitometry analysis of FLAG-MyD88 bands normalized to actin. (E) CLIP170 did not promote the ubiquitination of FLAG-MyD88. The ubiquitination assay was performed as described before with the FLAG-MyD88 as the substrate. Ubiquitination status of MyD88 was not affected by CLIP170. (F) CLIP170 induces degradation of endogenous TIRAP. RAW264 cells were transfected with EV or HA-CLIP170 followed by detection of endogenous TIRAP and MyD88 using anti-TIRAP and anti-MyD88 Abs, respectively. Actin served as the loading control. Immunoblots are representative of two independent experiments. EV, empty vector; IB, immunoblotting; IP, immunoprecipitation.

FIGURE 1.

CLIP170 induces ubiquitination and degradation of TIRAP. (A) CLIP170 interacts with TIRAP. HEK293T cells were cotransfected with equal amounts of HA-CLIP170 and FLAG-TIRAP plasmids. Twenty-four hours posttransfection, cells were lysed and FLAG-TIRAP was immunoprecipitated using anti-FLAG Ab followed by immunoblotting. The blot was probed with anti-HA Ab to detect the coimmunoprecipitated HA-CLIP170 followed by detection of FLAG-TIRAP by anti-FLAG Ab. The whole-cell lysates were also subjected to immunoblotting followed by immunodetection of HA-CLIP170 and FLAG-TIRAP. (B) CLIP170 promotes degradation of TIRAP. HEK293T cells were cotransfected with FLAG-TIRAP and increasing concentrations of HA-CLIP170 plasmids. Twenty-four hours posttransfection, cells were lysed and subjected to immunoblotting. The blot was probed with anti-FLAG and anti-HA Abs to detect FLAG-TIRAP and HA-CLIP170, respectively. Actin served as the loading control. The right panel of the immunoblot shows the densitometry analysis of FLAG-TIRAP bands normalized to actin. (C) CLIP170 induces ubiquitination of TIRAP. HEK293T cells were cotransfected with various combinations of FLAG-TIRAP, MYC-CLIP170, and HA-ubiquitin as indicated. Twenty-four hours posttransfection, cells were treated with 20 μM MG132 for 4 h as indicated in the figure. Cells were then lysed and FLAG-TIRAP was immunoprecipitated followed by immunoblotting. The blot was probed with anti-HA Ab to detect the HA-ubiquitin–conjugated FLAG-TIRAP. CLIP170 enhanced the ubiquitination of CLIP170 in MG132-treated or untreated cells. Similar concentrations of FLAG-TIRAP and MYC-CLIP170 resulted in accumulation of ubiquitinated FLAG-TIRAP in the immunoprecipitated samples. (D) CLIP170 did not induce degradation of MyD88. HEK293T cells were cotransfected with 300 ng of FLAG-MyD88 and increasing concentrations of HA-CLIP170. Twenty-four hours posttransfection, cells were lysed and subjected to immunoblotting. The blot was probed with anti-FLAG and anti-HA Abs to detect FLAG-MyD88 and HA-CLIP170, respectively. Degradation of MyD88 was not observed with increasing concentrations of HA-CLIP170. The right panel of the immunoblot shows the densitometry analysis of FLAG-MyD88 bands normalized to actin. (E) CLIP170 did not promote the ubiquitination of FLAG-MyD88. The ubiquitination assay was performed as described before with the FLAG-MyD88 as the substrate. Ubiquitination status of MyD88 was not affected by CLIP170. (F) CLIP170 induces degradation of endogenous TIRAP. RAW264 cells were transfected with EV or HA-CLIP170 followed by detection of endogenous TIRAP and MyD88 using anti-TIRAP and anti-MyD88 Abs, respectively. Actin served as the loading control. Immunoblots are representative of two independent experiments. EV, empty vector; IB, immunoblotting; IP, immunoprecipitation.

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Selective protein degradation in eukaryotic cells is achieved by ubiquitination of target proteins and their degradation by the 20S proteasome complex. Therefore, we sought to examine whether CLIP170 could promote the ubiquitination of TIRAP. To analyze this, we performed a ubiquitination assay by cotransfecting HEK293T cells with various combinations of FLAG-TIRAP, MYC-CLIP170, and HA-ubiquitin constructs as described in Fig. 1C. Twenty-four hours after the transfections, cells were treated with the irreversible proteasome inhibitor MG132 for 4 h to inhibit the degradation of ubiquitinated FLAG-TIRAP. Next, FLAG-TIRAP was immunoprecipitated, and conjugation of HA-ubiquitin to the FLAG-TIRAP was analyzed by immunoblotting using anti-HA Ab. Ubiquitinated TIRAP could be detected in MG132-treated or untreated cells, and the levels of TIRAP ubiquitination was dramatically increased in the presence of CLIP170 (Fig. 1C). This suggests that CLIP170 promotes ubiquitination of TIRAP.

TIRAP serves as the bridging adaptor for recruiting the signaling adaptor MyD88 to the plasma membrane–bound TLR2 and TLR4 (4). Therefore, we wanted to analyze whether CLIP170 promotes degradation of MyD88. To examine this, we cotransfected HEK293T cells with a constant amount of FLAG-MyD88 expression plasmid and increasing concentrations of HA-CLIP170 plasmid and they were sampled for immunoblot analysis. In contrast to a decrease in TIRAP overexpression upon increasing co-overexpression of CLIP170, the levels of MyD88 expression did not decrease but slightly increased following increasing CLIP170 co-overexpression (Fig. 1D). However, reverse transcriptase or qPCR analyses did not indicate altered mRNA levels of MyD88 with CLIP170 (Supplemental Fig. 1). To confirm the substrate specificity for the ubiquitin ligase–like property of CLIP170, we performed a ubiquitination assay with FLAG-MyD88. CLIP170 did not induce ubiquitination of MyD88, as an equal amount of ubiquitinated MyD88 was observed in the presence or absence of CLIP170 (Fig. 1E). The levels of MyD88 did not change in the MG132-treated or untreated cells, suggesting that MyD88 did not undergo proteasome-mediated degradation (Fig. 1E). Next, we analyzed the levels of endogenous TIRAP and MyD88 in the presence of CLIP170 in macrophages. RAW264 mouse macrophages were transfected with HA-CLIP170 or empty vector and analyzed for the levels of TIRAP and MyD88 by immunoblotting using specific Abs. We observed an enhanced degradation of endogenous TIRAP in the RAW cells transfected with HA-CLIP170 whereas MyD88 was not degraded (Fig. 1F). The immunoblot analysis of RAW cell lysates using anti-TIRAP Abs from various suppliers detected a band of ∼35 kDa, which was degraded upon overexpression of CLIP170 (Supplemental Fig. 2A–C). The experimental data suggest that CLIP170 targets TIRAP for degradation.

Next, we analyzed whether CLIP170 induces degradation of TLR2, TLR4, and TLR9. HEK293 cells were cotransfected with constant amounts of FLAG-tagged TLRs and increasing concentrations of MYC-CLIP170. We observed slight degradation of FLAG-TLR2 with increasing concentrations of MYC-CLIP170 (Supplemental Fig. 2D) whereas FLAG-TLR4 underwent slightly enhanced ubiquitination (Supplemental Fig. 2E). Levels of FLAG-TLR9 remained unchanged with increasing concentrations of MYC-CLIP170 (Supplemental Fig. 2F).

Ubiquitination involves attachment of ubiquitin to the lysine residues of the substrate protein that can result in monoubiquitination or polyubiquitination. In monoubiquitination, a single ubiquitin moiety is attached to the lysine residues of the substrate whereas in polyubiquitination K48- or K63-linked homotypic ubiquitin chains are attached to the substrate (30). We analyzed the nature of CLIP170-induced ubiquitination of TIRAP by a ubiquitination assay using mutant versions of ubiquitin. HEK293T cells were cotransfected with FLAG-TIRAP, MYC-CLIP170, and various mutants of HA-tagged ubiquitin, namely UBI-K48R, UBI-K48 only, UBI-K63R, UBI-K63 only, and UBI-7KR. The UBI-K48R cannot form K48-linked ubiquitin chains whereas UBI-K48 only mutant can form only K48-linked ubiquitin chains. Similarly, the UBI-K63R cannot form K63-linked ubiquitin chains whereas UBI-K63 only can form only K63-linked ubiquitin chains. In the UBI-7KR, all seven lysine residues in ubiquitin were mutated to arginine, which cannot form homotypic chains. All the mutants are capable of mono- or multiple monoubiquitination of target proteins. Interestingly, CLIP170 promoted conjugation of all of the mutant versions of ubiquitin, including UBI-7KR to TIRAP (Fig. 2A, 2B). Conjugation of UBI-7KR to TIRAP suggests that CLIP170 promotes the monoubiquitination of TIRAP. The smeared appearance of the TIRAP band with UBI-7KR suggests that TIRAP undergoes multiple monoubiquitinations in the presence of CLIP170. Similarly, the presence of high-molecular mass smears of TIRAP with other ubiquitin mutants, which could form homotypic chains, suggests that CLIP170 also promotes the polyubiquitination of TIRAP (Fig. 2A, 2B).

FIGURE 2.

TIRAP undergoes polyubiquitination and multiple monoubiquitinations in the presence of CLIP170. (A and B) HEK293T cells were cotransfected with FLAG-TIRAP, CLIP170, and various lysine to arginine mutants of HA-ubiquitin followed by immunoprecipitation of FLAG-TIRAP and immunoblotting. CLIP170 mediated conjugation of all the mutant versions of ubiquitin, including UBI7KR, where all the lysine residues are mutated to arginine. The vertical white lines indicate where the figure panels were joined.

FIGURE 2.

TIRAP undergoes polyubiquitination and multiple monoubiquitinations in the presence of CLIP170. (A and B) HEK293T cells were cotransfected with FLAG-TIRAP, CLIP170, and various lysine to arginine mutants of HA-ubiquitin followed by immunoprecipitation of FLAG-TIRAP and immunoblotting. CLIP170 mediated conjugation of all the mutant versions of ubiquitin, including UBI7KR, where all the lysine residues are mutated to arginine. The vertical white lines indicate where the figure panels were joined.

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CLIP170 regulates microtubule dynamics and has been implicated in stabilization of microtubules (31). We analyzed the subcellular localization of CLIP170 in MEF cells. MYC-CLIP170 was overexpressed in MEF cells followed by immunocytochemistry using anti–MYC-FITC to visualize CLIP170 (Supplemental Fig. 3A–C). CLIP170 induced dramatic microtubule bundling in MEF cells. As we demonstrated before, Brucella effector protein TcpB that interacted with CLIP170 also induced severe microtubule bundling and strongly colocalized with microtubules (Supplemental Fig. 3D–F).

TIRAP is indispensable for the MyD88-dependent pathway of TLR4 signaling, and, therefore, the negative regulation of this pathway can be achieved by selected elimination of TIRAP by proteins such as SOCS-1. Given that CLIP170 promotes ubiquitination and degradation of TIRAP, we wanted to examine whether CLIP170 affects the LPS-induced signaling mediated by TLR4. We performed luciferase reporter assays to analyze the effect of CLIP170 in TLR4-mediated NF-κB activation. HEK293 cells, stably expressing TLR4, CD14, and MD2, were cotransfected with reporter plasmids and increasing concentrations of CLIP170 followed by challenge with LPS and NF-κB reporter assay. The assay indicated that CLIP170 could efficiently suppress TLR4-induced NF-κB activation in a dose-dependent manner (Fig. 3A). To examine whether the action of CLIP170 is specific for TLR4, we analyzed the effect of CLIP170 on TLR9 signaling, which does not require TIRAP for transduction. HEK293T cells were cotransfected with murine TLR9 plasmid, reporter plasmids, and increasing concentrations of CLIP170 followed by induction with ODN and NF-κB reporter assay. CLIP170 did not affect the activation of NF-κB induced by TLR9 signaling (Fig. 3B). This suggests that CLIP170 targets the TLR4 signaling pathway where the role of TIRAP is indispensable.

FIGURE 3.

CLIP170 attenuates TLR4- but not TLR9-induced NF-κB activation. (A) HEK293 cells expressing human TLR4/CD14/MD2 were transfected with increasing concentrations of HA-CLIP170 (50, 100, and 250 ng), pNF-κB–Luc reporter plasmid (100 ng), and pRL-TK (50 ng). (B) For the TLR9 assay, HEK293T cells were cotransfected with mTLR9 (200 ng) plasmid along with other plasmids mentioned above. The total amount of DNA was made constant by adding empty vector. Twenty-four hours posttransfection, cells were challenged overnight with LPS (300 ng/ml) and ODN (1 μg/ml), respectively. Luciferase activity was measured using the Dual-Luciferase reporter assay system; the lower panels are immunoblots demonstrating the overexpression of HA-CLIP170. Actin was used as the loading control. (C) Overexpression of CLIP170 in mouse macrophages. J774 cells were transduced with lentiviral particles harboring CLIP170 or empty vector followed by immunoblotting and qPCR. The blot was probed with anti-CLIP170 Ab to examine the overexpression of CLIP170. Actin served as the loading control. (D) CLIP170 suppresses LPS-induced activation of IL-6 and TNF-α in mouse macrophages. J774 cells transduced with CLIP170 expression plasmid or empty vector were challenged with LPS (100 ng/ml) for the indicated time points, followed by ELISA and qPCR to quantify the levels of IL-6 and TNF-α. (EH) Effect of CLIP170 on Pam3CSK4-, ODN-, and TNF-α–induced proinflammatory cytokines and LPS-induced IFN-β secretion. CLIP170 weakly suppressed Pam3CSK4-induced IL-6 and TNF-α levels (E) whereas it did not affect ODN-induced (F) or TNF-α–induced (G) proinflammatory cytokines or LPS-induced IFN-β (h) levels. Data are presented as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 3.

CLIP170 attenuates TLR4- but not TLR9-induced NF-κB activation. (A) HEK293 cells expressing human TLR4/CD14/MD2 were transfected with increasing concentrations of HA-CLIP170 (50, 100, and 250 ng), pNF-κB–Luc reporter plasmid (100 ng), and pRL-TK (50 ng). (B) For the TLR9 assay, HEK293T cells were cotransfected with mTLR9 (200 ng) plasmid along with other plasmids mentioned above. The total amount of DNA was made constant by adding empty vector. Twenty-four hours posttransfection, cells were challenged overnight with LPS (300 ng/ml) and ODN (1 μg/ml), respectively. Luciferase activity was measured using the Dual-Luciferase reporter assay system; the lower panels are immunoblots demonstrating the overexpression of HA-CLIP170. Actin was used as the loading control. (C) Overexpression of CLIP170 in mouse macrophages. J774 cells were transduced with lentiviral particles harboring CLIP170 or empty vector followed by immunoblotting and qPCR. The blot was probed with anti-CLIP170 Ab to examine the overexpression of CLIP170. Actin served as the loading control. (D) CLIP170 suppresses LPS-induced activation of IL-6 and TNF-α in mouse macrophages. J774 cells transduced with CLIP170 expression plasmid or empty vector were challenged with LPS (100 ng/ml) for the indicated time points, followed by ELISA and qPCR to quantify the levels of IL-6 and TNF-α. (EH) Effect of CLIP170 on Pam3CSK4-, ODN-, and TNF-α–induced proinflammatory cytokines and LPS-induced IFN-β secretion. CLIP170 weakly suppressed Pam3CSK4-induced IL-6 and TNF-α levels (E) whereas it did not affect ODN-induced (F) or TNF-α–induced (G) proinflammatory cytokines or LPS-induced IFN-β (h) levels. Data are presented as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Close modal

Next, we examined whether the overexpression of CLIP170 attenuates proinflammatory cytokine secretion by macrophages. J774 cells were transduced with lentivirus harboring CLIP170 expression plasmid or empty vector, and the overexpression of CLIP170 was confirmed by immunoblotting using anti-CLIP170 Ab and qPCR (Fig. 3C). Next, lentivirus-transduced J774 cells were challenged with LPS and the levels of IL-6 and TNF-α were analyzed by ELISA and qPCR. J774 macrophages overexpressing CLIP170 secreted diminished levels of IL-6 and TNF-α compared with cells transduced with the empty vector (Fig. 3D). We did not observe a significant difference in TNF-α mRNA levels between CLIP170-overexpressed and control J774 cells (Fig. 3D). Next, we examined whether CLIP170 affects signaling from other TLR or non-TLR receptors. J774 cells that were transduced with CLIP170 expression plasmid were stimulated with Pam3CSK4 (Fig. 3E), ODN (Fig. 3F), and TNF-α (Fig. 3G). CLIP170 could weakly inhibit Pam3CSK4-mediated signaling (Fig. 3E) whereas ODN- or TNF-α–mediated signaling was not affected (Fig. 3F, 3G). LPS induces production of type I IFNs, which is mediated by the MyD88-independent pathway of TLR4 signaling (32). We did not observe suppression of LPS-induced IFN-β in J774 cells overexpressing CLIP170 (Fig. 3H). The experimental data suggest that CLIP170 mainly targets the MyD88-dependent pathway of TLR4 signaling.

As overexpression of CLIP170 attenuated TLR4-induced NF-κB activation and proinflammatory cytokine secretion, we wanted to examine whether silencing of endogenous CLIP170 leads to potentiation of proinflammatory cytokines. J774 cells were transduced with lentivirus harboring CLIP170-specific shRNA expression plasmid or control shRNA, and the downregulation of CLIP170 was confirmed by immunoblotting and qPCR (Fig. 4A). Next, CLIP170-silenced cells were challenged with LPS and levels of IL-6 and TNF-α were analyzed by qPCR and ELISA. CLIP170-silenced J774 cells expressed elevated levels of IL-6 compared with the cells transduced with scrambled shRNA (Fig. 4B). We observed significantly elevated levels of TNF-α only at the 1 h time point in CLIP170-silenced J774 cells (Fig. 4B). As noticed before, mRNA levels of TNF-α did not change significantly in CLIP170-silenced J774 cells in comparison with the control (Fig. 4B). Stimulation of CLIP170-silenced cells with Pam3CSK4 resulted in suppression of IL-6 and TNF-α at the early time points whereas no suppression was observed in ODN- or TNF-α–induced cells (Fig. 4C–E). As observed previously, IFN-β levels were not significantly altered in CLIP170-silenced cells compared with the control (Fig. 4F).

FIGURE 4.

Silencing of endogenous CLIP170 potentiates LPS-induced proinflammatory cytokines in macrophages. (A) shRNA-mediated knockdown of endogenous CLIP170 in mouse macrophages. J774 cells were transduced with lentiviral particles harboring CLIP170 shRNA or control shRNA expression plasmid followed by immunoblotting and qPCR analysis to examine the levels of endogenous CLIP170. The blot was probed with anti-CLIP170 Ab to detect the endogenous CLIP170. Actin served as the loading control. (B) Knockdown of endogenous CLIP170 potentiates LPS-induced expression of IL-6 and TNF-α in mouse macrophages. J774 cells transduced with CLIP170 shRNA expression plasmid or control shRNA plasmid were challenged with LPS (100 ng/ml) for the indicated time points, followed by ELISA and qPCR to quantify the levels of IL-6 and TNF-α. (CE) Expression of IL-6 and TNF-α in CLIP170-silenced J774 cells challenged with Pam3CSK4, ODN, and TNF-α. CLIP170 weakly suppressed Pam3CSK4-induced IL-6 and TNF-α levels (C) whereas it did not affect ODN-induced (D) or TNF-α– (E) induced proinflammatory cytokines. (F) Expression levels of IFN-β in CLIP170-silenced cells challenged with LPS. IFN-β levels were not significantly altered in CLIP170-silenced cells compared with control. Data are presented as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 4.

Silencing of endogenous CLIP170 potentiates LPS-induced proinflammatory cytokines in macrophages. (A) shRNA-mediated knockdown of endogenous CLIP170 in mouse macrophages. J774 cells were transduced with lentiviral particles harboring CLIP170 shRNA or control shRNA expression plasmid followed by immunoblotting and qPCR analysis to examine the levels of endogenous CLIP170. The blot was probed with anti-CLIP170 Ab to detect the endogenous CLIP170. Actin served as the loading control. (B) Knockdown of endogenous CLIP170 potentiates LPS-induced expression of IL-6 and TNF-α in mouse macrophages. J774 cells transduced with CLIP170 shRNA expression plasmid or control shRNA plasmid were challenged with LPS (100 ng/ml) for the indicated time points, followed by ELISA and qPCR to quantify the levels of IL-6 and TNF-α. (CE) Expression of IL-6 and TNF-α in CLIP170-silenced J774 cells challenged with Pam3CSK4, ODN, and TNF-α. CLIP170 weakly suppressed Pam3CSK4-induced IL-6 and TNF-α levels (C) whereas it did not affect ODN-induced (D) or TNF-α– (E) induced proinflammatory cytokines. (F) Expression levels of IFN-β in CLIP170-silenced cells challenged with LPS. IFN-β levels were not significantly altered in CLIP170-silenced cells compared with control. Data are presented as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Close modal

Next, we examined whether the silencing of endogenous CLIP170 in mice potentiated the LPS-induced inflammatory responses by employing an in vivo siRNA delivery technique. In vivo siRNA targeting CLIP170 or scrambled siRNA was complexed with the siRNA delivery agent Invivofectamine, followed by i.v. injection to 6-wk-old C57BL/6 female mice (50 μg of siRNA per mouse). To analyze the silencing of endogenous CLIP170, mice were sacrificed after 48 h of siRNA delivery followed by isolation of splenocytes and Kupffer cells. Subsequently, the endogenous levels of CLIP170 were analyzed in splenocytes and Kupffer cells by immunoblotting and qPCR. Both cell types displayed diminished levels of CLIP170 expression compared with the control, indicating the silencing of endogenous CLIP170 (Fig. 5A, 5B). We obtained a robust silencing of CLIP170 in the splenocytes (Fig. 5A). Next, we examined the levels of IL-6 and TNF-α in the splenocytes and liver of CLIP170-silenced or control mice. To determine the cytokine levels in splenocytes, isolated cells were challenged with LPS for various time points, followed by quantification of IL-6 and TNF-α by ELISA and qPCR. We observed elevated levels of both IL-6 and TNF-α in splenocytes from CLIP170-silenced mice (Fig. 5C). Next, we analyzed the levels of proinflammatory cytokines in the liver of CLIP170-silenced or control mice. Seventy-two hours after the siRNA delivery, mice were given sublethal injection of LPS (25 mg/kg) i.p. and liver was harvested after 2 h postinjection. Total RNA was isolated from a portion of the liver followed by cDNA synthesis and qPCR analysis to quantify the levels of IL-6 and TNF-α. We observed potentiated levels of IL-6 and TNF-α expression in the CLIP170-silenced mice compared with the control (Fig. 5D). The experimental data suggest that CLIP170 acts as the negative regulator of LPS-induced TLR4 signaling in mouse.

FIGURE 5.

Silencing of CLIP170 enhances LPS-induced proinflammatory cytokines in mice. (A and B) Depletion of CLIP170 in splenocytes or Kupffer cells derived from CLIP170 siRNA–treated mice. Mice were treated with CLIP170 siRNA or control siRNA for 48 h, followed by collection of splenocytes (A) or Kupffer cells (B). The cells were analyzed by immunoblotting and qPCR to examine the levels of endogenous CLIP170. The blot was probed with anti-CLIP170 to detect the endogenous CLIP170. Actin served as the loading control. (C) Depletion of CLIP170 potentiates the expression of IL-6 and TNF-α in splenocytes. The splenocytes isolated from mice injected with CLIP170-specific or control siRNA were challenged with LPS (100 ng/ml) for the indicated time points followed by quantification of IL-6 and TNF-α by ELISA and qPCR. (D) Potentiated levels of IL-6 and TNF-α in the liver of CLIP170-silenced mice. Seventy-two hours after the siRNA delivery, mice were injected with LPS (25 mg/kg) i.p. and the levels of IL-6 and TNF-α were quantified by qPCR. Data are presented as mean ± SD from two independent experiments of six mice per group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Silencing of CLIP170 enhances LPS-induced proinflammatory cytokines in mice. (A and B) Depletion of CLIP170 in splenocytes or Kupffer cells derived from CLIP170 siRNA–treated mice. Mice were treated with CLIP170 siRNA or control siRNA for 48 h, followed by collection of splenocytes (A) or Kupffer cells (B). The cells were analyzed by immunoblotting and qPCR to examine the levels of endogenous CLIP170. The blot was probed with anti-CLIP170 to detect the endogenous CLIP170. Actin served as the loading control. (C) Depletion of CLIP170 potentiates the expression of IL-6 and TNF-α in splenocytes. The splenocytes isolated from mice injected with CLIP170-specific or control siRNA were challenged with LPS (100 ng/ml) for the indicated time points followed by quantification of IL-6 and TNF-α by ELISA and qPCR. (D) Potentiated levels of IL-6 and TNF-α in the liver of CLIP170-silenced mice. Seventy-two hours after the siRNA delivery, mice were injected with LPS (25 mg/kg) i.p. and the levels of IL-6 and TNF-α were quantified by qPCR. Data are presented as mean ± SD from two independent experiments of six mice per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Negative regulators of TLR signaling are induced by the agonists of respective TLRs (33, 34). Because CLIP170 negatively regulates the TLR4 signaling, we examined whether CLIP170 expression is induced by the TLR4 ligand LPS. To analyze this, J774 cells were challenged with LPS followed by analysis of endogenous levels of CLIP170 mRNA and protein by qPCR and immunoblotting, respectively. We observed diminished levels of CLIP170 transcript and protein expression during the first 15 min of LPS induction compared with the 0 h time point (Supplemental Fig. 4). This is followed by a gradual increase of CLIP170 expression up to 2–3 h. It appears that CLIP170 expression was downregulated at the initial stages of TLR4 signaling followed by its upregulation at later stages. TLR4 signaling activates the transcription factor NF-κB, which in turn activates many genes that harbor NF-κB–responsive elements. Interestingly, experimentally validated NF-κB1 binding sites were reported in the promoter region of both mouse and human CLIP170 (Qiagen).

CLIP170 is multifunctional protein that is involved in important cellular processes, including intracellular transport and signaling. CLIP170 interacts with the Brucella effector protein TcpB that promotes ubiquitination and degradation of TIRAP to subvert TLR2 and TLR4 signaling (17). TcpB is unlikely to harbor a ubiquitin ligase property, and it may recruit host proteins for ubiquitination and degradation of TIRAP (17). In this study, we report that CLIP170 interacts with TIRAP and induces its ubiquitination and degradation. Human CLIP170 maps on the reverse strand of chromosome 12 at the 12q24.3 position, which spans 152.26 kb, and the locus is reported to be complex (35). The CLIP170 gene is predicted to transcribe 29 different mRNAs, 21 alternatively spliced variants, and 8 unspliced forms (35). The gene is highly expressed, 4.3-fold the average gene, and the RNA sequencing expression analysis indicated its expression in various tissues, including brain, skeletal muscle, liver, spleen, colon, lymph nodes, testes, and thymus (35, 36). Four isoforms of CLIP170, namely Restin, CLIP170, CLIP170 (11), and CLIP170 (11+35), have been cloned and analyzed from humans and chickens (3740). All four CLIP170 isoforms appear to carry the identical CAP-Gly domains at the N terminus and zinc knuckles at the C terminus. Structural analysis indicated that CLIP170 forms parallel dimers through its central α helical coiled-coil rod domain (23). The rod domain of CLIP170 has two kinks, which may contribute to the folded back state of CLIP170 that regulates the activity of the protein (41). The changes in the rod domain of CLIP170 by alternative splicing may regulate its activity and recruitment for diverse cellular functions.

Our analysis revealed a previously unrecognized ubiquitin ligase–like property of CLIP170. Interestingly, CLIP170 does not possess classical ubiquitin ligase domains such as HECT, RING, or U box (42). Therefore, CLIP170 may act as a scaffold to facilitate the ubiquitination of target proteins. Familial cylindromatosis tumor suppressor (CYLD) contains three CAP-Gly domains and inhibits NF-ĸB activation by cleaving the K63-linked polyubiquitin chains from the ubiquitin ligases, TNFR-associated factor-2, TNFR-associated factor-6, and NEMO (4345). In contrast, CLIP170 promoted ubiquitination of target proteins. SOCS1 suppresses TLR4 signaling through proteasome-mediated degradation of TIRAP (11). We assume that CLIP170 also employs a similar mechanism to attenuate TLR4 signaling. Our studies suggest that CLIP170 mediates conjugation of mono- and polyubiquitin to TIRAP. Polyubiquitination through K48 results in proteasome-mediated degradation of the target proteins (46, 47). Monoubiquitination has also been shown to direct the proteins for degradation (4850).

Signaling through TLR4 is heavily regulated owing to the extreme toxicity of TLR4 signaling (51). The regulators target multiple points of the complex TLR4 signaling pathway. We observed that the expression of CLIP170 in mouse macrophages was modulated by the TLR4 agonist LPS. A transient downregulation of CLIP170 was observed in the first 15 min of LPS induction followed by a steady increase of CLIP170 expression. TLR4 signaling occurs in two phases, an early phase mediated by the TLR4–MyD88–TIRAP complex and a late phase mediated by the TLR4–TIRAP–4-1BBL complex (52). Early phase may last for the first 15 min of LPS induction, as TIRAP degradation is initiated during this period (11). Subsequently, TIRAP may be resynthesized for the late phase signaling of TLR4 (52). Hence, in the early phase of TLR4 signaling, TIRAP degradation should be prevented to facilitate the unhindered signaling from TLR4. Therefore, it is conceivable that a transient downregulation of CLIP170 at the early stage of TLR4 signaling may ensure the availability of signal-competent TIRAP. Early TLR4 signaling may upregulate CLIP170 expression through NF-κB that in turn ubiquitinates and degrades TIRAP, resulting in the negative regulation of TLR4 signaling.

Overexpression of CLIP170 or TcpB induced severe microtubule bundling in various cell types. Taxol-stabilized microtubules have been reported to relieve the autoinhibition of CLIP170 (53). We previously demonstrated that TcpB mimics the properties of taxol by acting as a microtubule stabilization agent (14). Therefore, it is possible that the stabilization of microtubules by TcpB may activate the autoinhibited CLIP170 molecules. Besides this, the perturbation of microtubule dynamics by taxol or nocodazole was reported to cause dissociation of CLIP170 from the microtubule ends (54, 55). Hence, it can be speculated that TcpB-induced microtubule modulation may cause displacement of CLIP170 from the microtubule tips, which may facilitate the recruitment of CLIP170 for the suppression of TLR4 signaling.

In summary, we report an unexpected finding that CLIP170 negatively regulates TLR4 signaling by the targeted ubiquitination and degradation of TIRAP. Furthermore, we observed that CLIP170 expression is modulated by LPS to maintain the cellular homeostasis. The role of CLIP170 in TLR signaling and inflammation has not been recognized previously. However, CLIP170 has been implicated in disease conditions such as Hodgkin disease, anaplastic large cell lymphoma, and autosomal recessive intellectual disability (40, 56, 57). The role of CLIP170 has recently been reported in improving the sensitivity of paclitaxel in breast cancer cells (58). Our findings help to understand the underlying mechanisms of CLIP170-mediated disease pathogenesis, which may ultimately help to develop novel therapeutic interventions.

We thank G. Ramadevi and Shashikant Gawai for help with mice experiments and confocal microscopy, respectively. We thank Dr. Sathya Velmurugan for proofreading the manuscript.

Work in the laboratory of G.R. is supported by funding from the Department of Biotechnology, Ministry of Science and Technology, Government of India, through the National Institute of Animal Biotechnology, Hyderabad, India. Work in the laboratory of G.S. is funded by National Institutes of Health Grant R01-AI-073558. G.R. was awarded National Institutes of Health Grant R03AI101611 to initiate this study. P.J. acknowledges a research fellowship for doctoral studies from Innovation in Science Pursuit for Inspired Research, Department of Science and Technology, Government of India. S.M. acknowledges a postdoctoral fellowship from the University Grant Commission, Government of India.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CAP-Gly

cytoskeleton-associated protein glycine-rich

CLIP170

cytoplasmic linker protein 170

HA

hemagglutinin

HEK

human embryonic kidney

MEF

mouse embryonic fibroblast

ODN

oligodeoxynucleotide

qPCR

quantitative PCR

shRNA

short hairpin RNA

siRNA

small interfering RNA

SOCS-1

suppressor of cytokine signalling-1

TIR

Toll/IL-1 receptor

TIRAP

TIR domain–containing adaptor protein.

1
Akira
,
S.
,
K.
Takeda
,
T.
Kaisho
.
2001
.
Toll-like receptors: critical proteins linking innate and acquired immunity.
Nat. Immunol.
2
:
675
680
.
2
Miggin
,
S. M.
,
L. A.
O’Neill
.
2006
.
New insights into the regulation of TLR signaling.
J. Leukoc. Biol.
80
:
220
226
.
3
O’Neill
,
L. A.
,
A. G.
Bowie
.
2007
.
The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling.
Nat. Rev. Immunol.
7
:
353
364
.
4
Kagan
,
J. C.
,
R.
Medzhitov
.
2006
.
Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling.
Cell
125
:
943
955
.
5
Kawai
,
T.
,
S.
Akira
.
2006
.
TLR signaling.
Cell Death Differ.
13
:
816
825
.
6
Lowe
,
E. L.
,
T. M.
Doherty
,
H.
Karahashi
,
M.
Arditi
.
2006
.
Ubiquitination and de-ubiquitination: role in regulation of signaling by Toll-like receptors.
J. Endotoxin Res.
12
:
337
345
.
7
O’Neill
,
L. A.
2009
.
Regulation of signaling by non-degradative ubiquitination.
J. Biol. Chem.
284
:
8209
.
8
Pickart
,
C. M.
2001
.
Mechanisms underlying ubiquitination.
Annu. Rev. Biochem.
70
:
503
533
.
9
Chuang
,
T. H.
,
R. J.
Ulevitch
.
2004
.
Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors.
Nat. Immunol.
5
:
495
502
.
10
Fearns
,
C.
,
Q.
Pan
,
J. C.
Mathison
,
T. H.
Chuang
.
2006
.
Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding.
J. Biol. Chem.
281
:
34592
34600
.
11
Mansell
,
A.
,
R.
Smith
,
S. L.
Doyle
,
P.
Gray
,
J. E.
Fenner
,
P. J.
Crack
,
S. E.
Nicholson
,
D. J.
Hilton
,
L. A.
O’Neill
,
P. J.
Hertzog
.
2006
.
Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation.
Nat. Immunol.
7
:
148
155
.
12
Radhakrishnan
,
G. K.
,
Q.
Yu
,
J. S.
Harms
,
G. A.
Splitter
.
2009
.
Brucella TIR domain-containing protein mimics properties of the Toll-like receptor adaptor protein TIRAP.
J. Biol. Chem.
284
:
9892
9898
.
13
Radhakrishnan
,
G. K.
,
G. A.
Splitter
.
2010
.
Biochemical and functional analysis of TIR domain containing protein from Brucella melitensis.
Biochem. Biophys. Res. Commun.
397
:
59
63
.
14
Radhakrishnan
,
G. K.
,
J. S.
Harms
,
G. A.
Splitter
.
2011
.
Modulation of microtubule dynamics by a TIR domain protein from the intracellular pathogen Brucella melitensis.
Biochem. J.
439
:
79
83
.
15
Cirl
,
C.
,
A.
Wieser
,
M.
Yadav
,
S.
Duerr
,
S.
Schubert
,
H.
Fischer
,
D.
Stappert
,
N.
Wantia
,
N.
Rodriguez
,
H.
Wagner
, et al
.
2008
.
Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins.
Nat. Med.
14
:
399
406
.
16
Salcedo
,
S. P.
,
M. I.
Marchesini
,
H.
Lelouard
,
E.
Fugier
,
G.
Jolly
,
S.
Balor
,
A.
Muller
,
N.
Lapaque
,
O.
Demaria
,
L.
Alexopoulou
, et al
.
2008
.
Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1.
PLoS Pathog.
4
:
e21
.
17
Sengupta
,
D.
,
A.
Koblansky
,
J.
Gaines
,
T.
Brown
,
A. P.
West
,
D.
Zhang
,
T.
Nishikawa
,
S. G.
Park
,
R. M.
Roop
II
,
S.
Ghosh
.
2010
.
Subversion of innate immune responses by Brucella through the targeted degradation of the TLR signaling adapter, MAL.
J. Immunol.
184
:
956
964
.
18
Pierre
,
P.
,
J.
Scheel
,
J. E.
Rickard
,
T. E.
Kreis
.
1992
.
CLIP-170 links endocytic vesicles to microtubules.
Cell
70
:
887
900
.
19
Slep
,
K. C.
,
R. D.
Vale
.
2007
.
Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1.
Mol. Cell
27
:
976
991
.
20
Akhmanova
,
A.
,
C. C.
Hoogenraad
,
K.
Drabek
,
T.
Stepanova
,
B.
Dortland
,
T.
Verkerk
,
W.
Vermeulen
,
B. M.
Burgering
,
C. I.
De Zeeuw
,
F.
Grosveld
,
N.
Galjart
.
2001
.
Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts.
Cell
104
:
923
935
.
21
Coquelle
,
F. M.
,
M.
Caspi
,
F. P.
Cordelières
,
J. P.
Dompierre
,
D. L.
Dujardin
,
C.
Koifman
,
P.
Martin
,
C. C.
Hoogenraad
,
A.
Akhmanova
,
N.
Galjart
, et al
.
2002
.
LIS1, CLIP-170’s key to the dynein/dynactin pathway.
Mol. Cell. Biol.
22
:
3089
3102
.
22
Holzbaur
,
E. L.
,
J. A.
Hammarback
,
B. M.
Paschal
,
N. G.
Kravit
,
K. K.
Pfister
,
R. B.
Vallee
.
1991
.
Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. [Published erratum appears in 1992 Nature 360: 695.]
Nature
351
:
579
583
.
23
Lansbergen
,
G.
,
Y.
Komarova
,
M.
Modesti
,
C.
Wyman
,
C. C.
Hoogenraad
,
H. V.
Goodson
,
R. P.
Lemaitre
,
D. N.
Drechsel
,
E.
van Munster
,
T. W.
Gadella
Jr.
, et al
.
2004
.
Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization.
J. Cell Biol.
166
:
1003
1014
.
24
Brunner
,
D.
,
P.
Nurse
.
2000
.
CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast.
Cell
102
:
695
704
.
25
Dujardin
,
D.
,
U. I.
Wacker
,
A.
Moreau
,
T. A.
Schroer
,
J. E.
Rickard
,
J. R.
De Mey
.
1998
.
Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment.
J. Cell Biol.
141
:
849
862
.
26
Komarova
,
Y. A.
,
A. S.
Akhmanova
,
S.
Kojima
,
N.
Galjart
,
G. G.
Borisy
.
2002
.
Cytoplasmic linker proteins promote microtubule rescue in vivo.
J. Cell Biol.
159
:
589
599
.
27
Tanenbaum
,
M. E.
,
N.
Galjart
,
M. A.
van Vugt
,
R. H.
Medema
.
2006
.
CLIP-170 facilitates the formation of kinetochore-microtubule attachments.
EMBO J.
25
:
45
57
.
28
Watson
,
P.
,
D. J.
Stephens
.
2006
.
Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells.
J. Cell Sci.
119
:
2758
2767
.
29
Li
,
P. Z.
,
J. Z.
Li
,
M.
Li
,
J. P.
Gong
,
K.
He
.
2014
.
An efficient method to isolate and culture mouse Kupffer cells.
Immunol. Lett.
158
:
52
56
.
30
Kerscher
,
O.
,
R.
Felberbaum
,
M.
Hochstrasser
.
2006
.
Modification of proteins by ubiquitin and ubiquitin-like proteins.
Annu. Rev. Cell Dev. Biol.
22
:
159
180
.
31
Pierre
,
P.
,
R.
Pepperkok
,
T. E.
Kreis
.
1994
.
Molecular characterization of two functional domains of CLIP-170 in vivo.
J. Cell Sci.
107
:
1909
1920
.
32
Akira
,
S.
,
K.
Takeda
.
2004
.
Toll-like receptor signalling.
Nat. Rev. Immunol.
4
:
499
511
.
33
Kinjyo
,
I.
,
T.
Hanada
,
K.
Inagaki-Ohara
,
H.
Mori
,
D.
Aki
,
M.
Ohishi
,
H.
Yoshida
,
M.
Kubo
,
A.
Yoshimura
.
2002
.
SOCS1/JAB is a negative regulator of LPS-induced macrophage activation.
Immunity
17
:
583
591
.
34
Kobayashi
,
K.
,
L. D.
Hernandez
,
J. E.
Galán
,
C. A.
Janeway
Jr.
,
R.
Medzhitov
,
R. A.
Flavell
.
2002
.
IRAK-M is a negative regulator of Toll-like receptor signaling.
Cell
110
:
191
202
.
35
Thierry-Mieg
,
D.
,
J.
Thierry-Mieg
.
2006
.
AceView: a comprehensive cDNA-supported gene and transcripts annotation.
Genome Biol.
7
(
Suppl. 1
):
S12.11
S12.14
.
36
Pipes
,
L.
,
S.
Li
,
M.
Bozinoski
,
R.
Palermo
,
X.
Peng
,
P.
Blood
,
S.
Kelly
,
J. M.
Weiss
,
J.
Thierry-Mieg
,
D.
Thierry-Mieg
, et al
.
2013
.
The non-human primate reference transcriptome resource (NHPRTR) for comparative functional genomics.
Nucleic Acids Res.
41
(
database issue
):
D906
D914
.
37
Griparic
,
L.
,
T. C.
Keller
III
.
1999
.
Differential usage of two 5′ splice sites in a complex exon generates additional protein sequence complexity in chicken CLIP-170 isoforms.
Biochim. Biophys. Acta
1449
:
119
124
.
38
Griparic
,
L.
,
T. C.
Keller
.
1998
.
Identification and expression of two novel CLIP-170/Restin isoforms expressed predominantly in muscle.
Biochim. Biophys. Acta
1405
:
35
46
.
39
Griparic
,
L.
,
J. M.
Volosky
,
T. C.
Keller
III
.
1998
.
Cloning and expression of chicken CLIP-170 and restin isoforms.
Gene
206
:
195
208
.
40
Bilbe
,
G.
,
J.
Delabie
,
J.
Brüggen
,
H.
Richener
,
F. A.
Asselbergs
,
N.
Cerletti
,
C.
Sorg
,
K.
Odink
,
L.
Tarcsay
,
W.
Wiesendanger
, et al
.
1992
.
Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin’s disease.
EMBO J.
11
:
2103
2113
.
41
Scheel
,
J.
,
P.
Pierre
,
J. E.
Rickard
,
G. S.
Diamantopoulos
,
C.
Valetti
,
F. G.
van der Goot
,
M.
Häner
,
U.
Aebi
,
T. E.
Kreis
.
1999
.
Purification and analysis of authentic CLIP-170 and recombinant fragments.
J. Biol. Chem.
274
:
25883
25891
.
42
Deshaies
,
R. J.
,
C. A.
Joazeiro
.
2009
.
RING domain E3 ubiquitin ligases.
Annu. Rev. Biochem.
78
:
399
434
.
43
Trompouki
,
E.
,
E.
Hatzivassiliou
,
T.
Tsichritzis
,
H.
Farmer
,
A.
Ashworth
,
G.
Mosialos
.
2003
.
CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members.
Nature
424
:
793
796
.
44
Kovalenko
,
A.
,
C.
Chable-Bessia
,
G.
Cantarella
,
A.
Israël
,
D.
Wallach
,
G.
Courtois
.
2003
.
The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination.
Nature
424
:
801
805
.
45
Brummelkamp
,
T. R.
,
S. M.
Nijman
,
A. M.
Dirac
,
R.
Bernards
.
2003
.
Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB.
Nature
424
:
797
801
.
46
Hicke
,
L.
2001
.
Protein regulation by monoubiquitin.
Nat. Rev. Mol. Cell Biol.
2
:
195
201
.
47
Hochstrasser
,
M.
1996
.
Ubiquitin-dependent protein degradation.
Annu. Rev. Genet.
30
:
405
439
.
48
Boutet
,
S. C.
,
S.
Biressi
,
K.
Iori
,
V.
Natu
,
T. A.
Rando
.
2010
.
Taf1 regulates Pax3 protein by monoubiquitination in skeletal muscle progenitors.
Mol. Cell
40
:
749
761
.
49
Rott
,
R.
,
R.
Szargel
,
J.
Haskin
,
R.
Bandopadhyay
,
A. J.
Lees
,
V.
Shani
,
S.
Engelender
.
2011
.
α-Synuclein fate is determined by USP9X-regulated monoubiquitination.
Proc. Natl. Acad. Sci. USA
108
:
18666
18671
.
50
Yin
,
H.
,
Y.
Gui
,
G.
Du
,
M. A.
Frohman
,
X. L.
Zheng
.
2010
.
Dependence of phospholipase D1 multi-monoubiquitination on its enzymatic activity and palmitoylation.
J. Biol. Chem.
285
:
13580
13588
.
51
Liew
,
F. Y.
,
D.
Xu
,
E. K.
Brint
,
L. A.
O’Neill
.
2005
.
Negative regulation of Toll-like receptor-mediated immune responses.
Nat. Rev. Immunol.
5
:
446
458
.
52
Ma
,
J.
,
B. R.
Bang
,
J.
Lu
,
S. Y.
Eun
,
M.
Otsuka
,
M.
Croft
,
P.
Tobias
,
J.
Han
,
O.
Takeuchi
,
S.
Akira
, et al
.
2013
.
The TNF family member 4-1BBL sustains inflammation by interacting with TLR signaling components during late-phase activation.
Sci. Signal.
6
:
ra87
.
53
Weisbrich
,
A.
,
S.
Honnappa
,
R.
Jaussi
,
O.
Okhrimenko
,
D.
Frey
,
I.
Jelesarov
,
A.
Akhmanova
,
M. O.
Steinmetz
.
2007
.
Structure-function relationship of CAP-Gly domains.
Nat. Struct. Mol. Biol.
14
:
959
967
.
54
Perez
,
F.
,
G. S.
Diamantopoulos
,
R.
Stalder
,
T. E.
Kreis
.
1999
.
CLIP-170 highlights growing microtubule ends in vivo.
Cell
96
:
517
527
.
55
Dragestein
,
K. A.
,
W. A.
van Cappellen
,
J.
van Haren
,
G. D.
Tsibidis
,
A.
Akhmanova
,
T. A.
Knoch
,
F.
Grosveld
,
N.
Galjart
.
2008
.
Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.
J. Cell Biol.
180
:
729
737
.
56
Larti
,
F.
,
K.
Kahrizi
,
L.
Musante
,
H.
Hu
,
E.
Papari
,
Z.
Fattahi
,
N.
Bazazzadegan
,
Z.
Liu
,
M.
Banan
,
M.
Garshasbi
, et al
2014
.
A defect in the CLIP1 gene (CLIP-170) can cause autosomal recessive intellectual disability.
Eur J Hum Genet.
23
:
331
336
.
57
Delabie
,
J.
,
R.
Shipman
,
J.
Brüggen
,
B.
De Strooper
,
F.
van Leuven
,
L.
Tarcsay
,
N.
Cerletti
,
K.
Odink
,
V.
Diehl
,
G.
Bilbe
, et al
.
1992
.
Expression of the novel intermediate filament-associated protein restin in Hodgkin’s disease and anaplastic large-cell lymphoma.
Blood
80
:
2891
2896
.
58
Sun
,
X.
,
D.
Li
,
Y.
Yang
,
Y.
Ren
,
J.
Li
,
Z.
Wang
,
B.
Dong
,
M.
Liu
,
J.
Zhou
.
2012
.
Microtubule-binding protein CLIP-170 is a mediator of paclitaxel sensitivity.
J. Pathol.
226
:
666
673
.

The authors have no financial conflicts of interest.

Supplementary data