Protein Tyrosine Phosphatase 1B Is a Key Regulator of IFNAR1 Endocytosis and a Target for Antiviral Therapies

Protein tyrosine phosphatase 1B is a key regulator of IFNAR1 endocytosis and a target for antiviral therapies

Christopher J. Carbonea,1, Hui Zhenga,1, Sabyasachi Bhattacharyaa, John R. Lewisb, Alexander M. Reiterb, Paula Henthornb, Zhong-Yin Zhangc, Darren P. Bakerd, Radha Ukkiramapandiane, Kendra K. Bencea, and Serge Y. Fuchsa,2

aMari Lowe Comparative Oncology Center and Department of Animal Biology and bDepartment of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; cDepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202; dBiogen-IDEC, Cambridge, MA 02142; and eBest West Laboratories, Inc., Salt Lake City, UT 84115

Edited by David E. Levy, New York University School of Medicine, New York, NY, and accepted by the Editorial Board October 4, 2012 (received for review July 6, 2012)

Type 1 interferons (IFN1) elicit antiviral defenses by activating the ubiquitination (12) and that TYK2 knockout mice (whose IFNAR1 cognate receptor composed of IFN-α/β receptor chain 1 (IFNAR1) and lacks an analogous motif) display normal levels of IFNAR1 (16, 17). IFNAR2. Down-regulation of this receptor occurs through IFN1-stim- In human cells, unmasking of Y466 and its interaction with AP2 is stimulated by IFNAR1 ubiquitination (12) facilitated by the ulated IFNAR1 ubiquitination, which exposes a Y466-based linear β endocytic motif within IFNAR1 to recruitment of the adaptin pro- -Trcp E3 ubiquitin ligase, which is recruited upon phosphorylation of Ser-535 within the IFNAR1 degron (18, 19). Such phosphory- tein-2 complex (AP2) and ensuing receptor endocytosis. Paradoxi- lation could be induced by IFN-α/β and mediated by activities of cally, IFN1-induced Janus kinase-mediated phosphorylation of Y466 JAK (20, 21) and protein kinase D2 (22). Alternatively, a basal fi is expected to decrease its af nity for AP2 and to inhibit the endo- phosphorylation of Ser-535 by casein kinase 1α (23) can be stim- cytic rate. To explain how IFN1 promotes Y466 phosphorylation yet ulated by numerous inducers of ligand-independent IFNAR1 stimulates IFNAR1 internalization, we proposed that the activity of a ubiquitination (20). These inducers—including activators of protein tyrosine phosphatase (PTP) is required to enable both events pathogen recognition receptors (24), the unfolded protein re- by dephosphorylating Y466. An RNAi-based screen identified PTP1B sponse (25), or proinflammatory cytokines such as interleukin-1 as a specific regulator of IFNAR1 endocytosis stimulated by IFN1, but (IL-1) (26, 27)—act via p38 kinase-dependent priming phos- not by ligand-independent inducers of IFNAR1 ubiquitination. PTP1B phorylation that does not require JAK activity (28, 29). Both li- is a promising target for treatment of obesity and diabetes; numer- gand/JAK-dependent and -independent pathways promote fi IFNAR1 ubiquitination, endocytosis, and degradation and restrict ous research programs are aimed at identi cation and characteriza- H tion of clinically relevant inhibitors of PTP1B. PTP1B is capable of the extent of IFN1 signaling (reviewed in ref. 30; see Fig. 4 ). Our current work was triggered by an unexpected observation that, binding and dephosphorylating IFNAR1. Genetic or pharmacologic under conditions when IFN-α or IL-1β induced comparable increa- modulation of PTP1B activity regulated IFN1 signaling in a manner ses in IFNAR1 ubiquitination, the rate of IFNAR1 endocytosis and dependent on the integrity of Y466 within IFNAR1 in human cells. degradation was lower in IFN-α–treated cells. Indeed, the Y466- These effects were less evident in mouse cells whose IFNAR1 lacks an dependent mechanism of IFN-α–stimulated IFNAR1 endocytosis analogous motif. PTP1B inhibitors robustly augmented the antiviral appeared counterintuitive given an existing paradigm suggesting that effects of IFN1 against vesicular stomatitis and hepatitis C viruses in this residue has to remain unphosphorylated to efficiently bind AP2 human cells and proved beneficial in feline stomatitis patients. The and enable endocytosis (31), and a report that Y466 undergoes JAK- clinical significance of these findings in the context of using PTP1B mediated phosphorylation in response to IFN-α (32). To explain inhibitors to increase the therapeutic efficacy of IFN against viral how IFN-α promotes Y466 phosphorylation yet stimulates IFNAR1 infections is discussed. endocytosis, we proposed that the activity of a putative phosphatase enables both events by dephosphorylating Y466. α β Here we describe the results of an RNAi-based screen that iden- ype 1 interferons (IFN1, including IFN- / ) are widely used tified protein tyrosine phosphatase 1B (PTP1B) as a specificregu- Tto treat patients with viral infections (1–5). These cytokines lator of IFNAR1 endocytosis and thereby IFN1 signaling in human, elicit their antiviral effects by inducing IFN-stimulated genes (6, 7) but not in mouse, cells. PTP1B is known as an important regulator of whose transcription is activated as a result of a signal transduction intracellular signaling (33). PTP1B inhibitors are being sought to pathway involving binding of IFN1 to its receptor [consisting of regulate leptin and insulin signaling and, accordingly, function as IFN-α/β receptor chain 1 (IFNAR1) and IFNAR2] followed by potential therapeutics for the treatment of type 2 diabetes mellitus activation of Janus kinases (JAK; TYK2 and JAK1). These kinases and obesity (34–37). Novel, potent, selective, and orally available induce tyrosine phosphorylation of signal transducers and activa- inhibitors of this enzyme were recently reported (34, 38, 39). We tors of transcription (STAT1/2) and formation of transcriptionally demonstrate that PTP1B regulates IFN1 signaling in human cells and active complexes that recognize IFN-stimulated regulatory ele- that PTP1B inhibitors robustly synergize with IFN1 to protect human cellsagainsthepatitisCvirus(HCV) and vesicular stomatitis virus ments (ISRE) within the IFN-stimulated genes, the products of (VSV), and elicit a therapeutic effect in cats suffering from feline which suppress viral replication and stimulate immune responses stomatitis. The clinical significance of these findings is discussed. (reviewed in refs. 8–10). The initial sensitivity of cells to IFN1 depends on cell surface receptor density that is regulated by endocytosis and subsequent Author contributions: C.J.C., H.Z., S.B., J.R.L., A.M.R., P.H., Z.-Y.Z., D.P.B., R.U., K.K.B., and lysosomal degradation (11). In human cells, endocytosis of this S.Y.F. designed research; C.J.C., H.Z., S.B., J.R.L., A.M.R., P.H., R.U., K.K.B., and S.Y.F. per- receptor is mediated by the interaction between the adaptin pro- formed research; Z.-Y.Z., D.P.B., R.U., and K.K.B. contributed new reagents/analytic tools; tein-2 complex (AP2) endocytic machinery complex and the tyro- C.J.C., H.Z., S.B., J.R.L., A.M.R., P.H., Z.-Y.Z., D.P.B., R.U., K.K.B., and S.Y.F. analyzed data; sine (Y466)-based linear endocytic motif within the IFNAR1 subunit and C.J.C., H.Z., J.R.L., A.M.R., Z.-Y.Z., D.P.B., K.K.B., and S.Y.F. wrote the paper. (12). Such interaction is generally obscured by the IFNAR1-associ- Conflict of interest statement: D.P.B. is an employee and stockholder of Biogen Idec. ated TYK2 kinase (13); accordingly, human cells lacking TYK2 This article is a PNAS Direct Submission. D.E.L. is a guest editor invited by the Editorial Board. exhibit a robust basal endocytosis and degradation of IFNAR1 (14, 1C.J.C. and H.Z. contributed equally to this work. 15) as long as integrity of the Y466-based motif is preserved (13). 2To whom correspondence should be addressed. E-mail: [email protected]. Importance of this motif is further highlighted by reports that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the human Y466F mutant is poorly endocytosed despite a robust 1073/pnas.1211491109/-/DCSupplemental.

19226–19231 | PNAS | November 20, 2012 | vol. 109 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1211491109 Downloaded by guest on September 28, 2021 Results efficacy of IL-1β–induced endocytosis remained intact (Fig. S1C). The proteolytic turnover of IFNAR1 [assessed by a cycloheximide Endogenous PTP1B and IFNAR1 could be coimmunoprecipitated (CHX) chase assay] was more robustly stimulated by IL-1β than from the lysates of cells treated with IFN1 (Fig. 1D and Fig. S1D), by IFN-α (Fig. 1A). As expected, IFN-α, but not IL-1β, stimulated indicating that PTP1B is capable of interacting with IFNAR1. phosphorylation of IFNAR1 on tyrosines (Fig. 1B). Given that Pretreatment of cells with pharmacological inhibitors of PTP1B treatment with either IL-1β or IFN-α led to a comparable increase such as CinnGel2Mel and sodium stibogluconate noticeably in- in Ser-535 phosphorylation and ubiquitination of IFNAR1 (Fig. hibited internalization of IFNAR1 in cells stimulated with IFN-α 1B), differences between efficacy of these two inducers in accel- (Fig. 1E). Similar results were obtained using two other inhibitors, erating IFNAR1 degradation was likely conferred by events that including a selective ZYZ inhibitor (referred to as Compound II in occur downstream of IFNAR1 ubiquitination. ref. 41) and a recently described potent and bioavailable derivative As ubiquitination of human IFNAR1 promotes its internalization of cyano bromo naphthalene phosphonic acid (7-CBNP; referred to by unmasking the Y466-based linear endocytic motif for its in- as compound 3g in ref. 42) (Fig. S1E). A lesser extent of inhibition teraction with AP2 (12), we next analyzed the kinetics of IFNAR1 was achieved in cells treated with a pharmacologic inhibitor of endocytosis. Consistent with published results (12, 19), treatment of CD45 phosphatase (Fig. S1E). Overexpression of wild-type PTP1B cells with IFN-α increased the initial rate of IFNAR1 internalization noticeably augmented the IFNAR1 endocytic rate, whereas ex- as observed with either endogenous (Fig. 1C) or exogenously pression of the catalytically deficient substrate-trapping mutant expressed (Fig. S1A) receptor. Intriguingly, treatment of cells with PTP1BD181A (43) inhibited IFNAR1 endocytosis (Fig. 1F). Fur- IFN-α was noticeably less efficient at stimulating endocytosis of thermore, shRNA-mediated knockdown of PTP1B slowed down endogenous IFNAR1 than treatment with IL-1β (Fig. 1C). Given the initial internalization of IFNAR1 (Fig. 1G). These results col- that a Y466F mutation within the linear endocytic motif rendered lectively suggest that PTP1B functions as a positive regulator of the IFNAR1 relatively resistant to an increase in its internalization rate ligand-induced IFNAR1 endocytosis. in response to either IFN-α (Fig. S1A)orIL-1β (Fig. S1B), it is PTP1B is known to catalyze the dephosphorylation of nu- plausible that the putative postubiquitination event(s) that confers merous substrates (44–48), and its effect on IFNAR1 endocytosis differential capability of these inducers to stimulate IFNAR1 in- may be indirectly mediated by events upstream of unmasking ternalization occurs either upstream or at the level of Y466 expo- Y466 or downstream of AP2 recruitment to IFNAR1. However, sure. Because Y466 phosphorylation would be expected to decrease contrary to these possibilities, overexpression of PTP1B did not its affinity for AP2 (31), a lower efficacy of IFN-α as an endocytic efficiently increase the initial rate of internalization of the stimulator might be attributed to a differential ability to activate IFNAR1Y466F mutant (Fig. 2A). Given that this mutant was able to JAK and cause IFNAR1 Tyr phosphorylation (as seen in Fig. 1B). bind to PTP1B with efficacy similar to that of wild-type IFNAR1 Furthermore, the fact that IFN-α still stimulated IFNAR1 in- (Fig. 2B), it is likely that PTP1B, at least in part, regulates IFNAR1 ternalization suggested the existence of a putative PTP, the function endocytosis thorough an Y466-dependent mechanism. Consistent of which is to dephosphorylate Y466 and enable endocytosis of with this hypothesis, overexpression of PTP1B did not robustly the receptor. increase IFNAR1 endocytosis in cells that received RNAi against To identify such a phosphatase, we carried out a limited RNAi AP2 (Fig. 2C). These results strongly suggest that regulation of screen targeted against human classical PTPs (as classified in ref. IFNAR1 endocytosis by PTP1B is dependent on Y466 and AP2. 40). Cells transfected with RNAi against PTP1B exhibited a de- In line with coimmunoprecipitation analyses (Figs. 1D and 2B crease in IFN-α–stimulated endocytosis of IFNAR1, whereas the and Fig. S1D), the interaction between PTP1B (expressed in BIOCHEMISTRY

Fig. 1. PTP1B regulates the ligand-inducible endocytosis of IFNAR1. (A) Degradation of IFNAR1 in human 293-IL-1RI cells treated with CHX (20 μg/mL) and either PBS, IFN-α (1,000 U/mL), or IL-1β (2 ng/mL) for indicated times. (B) Phosphorylation and ubiquitination of IFNAR1 immunopuriﬁed from 293-IL-1RI cells treated with PBS, IFN-α (1,000 U/mL), or IL-1β (2 ng/ mL) for 10 min were analyzed by immunoblotting using the indicated antibodies. (C) Internalization rate of endogenous IFNAR1 in cells treated with PBS (diamonds), IFN-α (1,000 U/mL; squares), or IL-1β (2 ng/mL; circles) for indicated times was assessed from three independent experiments (each in six repli- cates) and depicted as average + SEM. (D) The interaction between endogenous IFNAR1 and PTP1B in 293-IL-1RI cells stimulated with either IFN-α (1,000 U/mL) or IL-1β (2 ng/mL) for 10 min was assessed by immunoprecipitation–immunoblotting using indicated antibodies. Levels of PTP1B in whole cell lysates (WCLs) were also analyzed. (E) Internalization of endogenous IFNAR1 in 293-IL-1RI cells treated with IFN- α (1,000 U/mL) in the presence of vehicle (DMSO; gray diamonds) or PTP1B inhibitors CinnGel2Me (10 μm; black triangles) or sodium stibogluconate (SSG; 10 μg/ mL; black squares). (F) Internalization of endogenous IFNAR1 in IFN-α–treated 293-IL-1RI cells transfected with empty vector (Vec; gray diamonds) or constructs for expression of PTP1B (WT, black squares; D181A mutant, open circles). (G) Internalization of endogenous IFNAR1 in IFN-α–treated 293-IL-1RI cells that received control shRNA (black diamonds) or shRNA against PTP1B (shP1B; gray squares). Inset shows the effect of these shRNAs on the levels of PTP1B (Upper) or β-actin (Lower).

Carbone et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19227 Downloaded by guest on September 28, 2021 To determine whether the latter reflects the properties of receptor or cells, we coexpressed human IFNAR2 with human IFNAR1 (wild-type or Y466F) in MEFs and treated them with species-specific IFN1. Internalization of the wild-type human IFNAR1 (but not of Y466F mutant) was increased upon coexpression of PTP1B. This effect was ligand-specific and not evident in the presence of mouse IFN1 (Fig. S1G), suggesting that presence of functional Y466 within IFNAR1 largely determines the extent of receptor endocytosis and its regulation by PTP1B even in mouse cells. To further test this possibility, we generated mouse–human chimeric IFNAR1 proteins (Fig. S2A) whose expression enabled MEFs from IFNAR1 knockout mice to respond to murine IFN1 (Fig. S2B) and rendered receptor endocytosis sensitive to a PTP1B inhibitor (Fig. S2C). In this model, coexpression of PTP1B stimulated the endocytosis of chimeric IFNAR1 as long as the integrity of Y466 was preserved (Fig. 3E). Finally, we used CHX assays to compare the turnover rate of various IFNAR1 proteins expressed in MEFs from wild-type or PTP1B knockout mice. Whereas kinetics of degradation of either murine IFNAR1 or chimeric mouse–human IFNAR1Y466F mutant was comparable, the chimeric receptor harboring intact Y466 was noticeably more stable in PTP1B-null cells (Fig. 3F and Fig. S2D). Collec- tively, these data suggest that PTP1B regulates IFNAR1 stability in a manner that largely depends on the integrity of a Tyr-based Fig. 2. PTP1B regulates Tyr phosphorylation and protein stability of IFNAR1 endocytic motif. in human cells. (A) Internalization of FLAG–IFNAR1 (WT, diamonds; or Y466F If the latter conclusion is correct, it would be expected that the role mutant, squares) in IFN-α–treated 293T cells that were cotransfected with of PTP1B in regulating the sensitivity to IFN1 should be much more empty vector (open symbols) or PTP1B (filled symbols). (B) Interaction be- important in human cells than in those of mouse origin. To test this tween endogenous PTP1B and FLAG–IFNAR1 expressed in 293T cells treated possibility, we determined the transactivation of ISRE-driven lucif- with IFN-α was determined by immunoprecipitation–immunoblotting using erase in MEFs transfected with human IFNAR2 and various com- the indicated antibodies. Loading was normalized to achieve comparable binations of human IFNAR1 and PTP1B (similar to the setup of levels of FLAG–IFNAR1 proteins. (C) Internalization of endogenous IFNAR1 in experiments shown in Fig. S1G). Without human IFNAR1, these IFN-α–treated 293T cells that received RNAi against AP2 (squares) or against cells could respond to mouse IFN-β, but not to human IFN-α (Fig. 4A luciferase (siCON; diamonds) and were cotransfected with either empty and Fig. S2E). Expression of human IFNAR1 (wild type or Y466F vector (open symbols) or PTP1B (filled symbols). (D) The interaction between mutant) conferred cell responsiveness to human IFN-α. Importantly, bacterially produced GST-tagged PTP1B proteins (WT or D181A mutant) with PTP1B overexpression significantly inhibited ISRE activation in cells FLAG-tagged IFNAR1 (WT or Y466F mutant) expressed in cells treated or not harboring wild-type, but not the Y466F mutant of, human IFNAR1 with IFN-α as indicated were analyzed by pulldown (using GSH beads) and (Fig. 4A and Fig. S2E). Given that the sensitivity to mouse IFN-β – immunoblotting. Normalized input levels of FLAG IFNAR1 in the cell lysates remained largely unaffected, these results suggest that PTP1B used for binding experiments are also shown. (E) In vitro Tyr dephosphorylation α fi α functions to attenuate IFN- signaling in a manner dependent on the of FLAG-tagged IFNAR1 puri ed from 293T cells treated or not with IFN- upon presence of human IFNAR1 and integrity of Y466. Importantly, the the incubation with indicated bacterially produced GST-tagged protein (for knockdown of PTP1B in human cells noticeably augmented ISRE 30 min at 37 °C) was analyzed by using the anti-phospho-Tyr antibody. activity conferred by endogenously produced IFN or stimulated by exogenously added ligand (Fig. 4B), further indicating that PTP1B negatively regulates the extent of responses to human IFN1. Coex- bacteria) and recombinant IFNAR1 purified from human cells D pression of PTP1B suppressed ISRE activities in IFNAR1-null treated with IFN1 was detected (Fig. 2 ). Importantly, an inactive MEFs harboring chimeric mouse–human IFNAR1 but not a chi- PTP1BD181A mutant was capable of efficiently trapping IFNAR1 C D meric Y466F mutant (Fig. 4 ), further emphasizing the importance as a putative substrate (Fig. 2 ). In turn, incubation with wild- of Y466 status in regulation of IFN1 signaling by PTP1B. type, but not D181A mutant, PTP1B protein led to a decreased We next determined the role of PTP1B in modulating IFN- phospho-Tyr signal on IFNAR1 purified from IFN1-treated cells α–induced cellular defenses against HCV in human Huh7.5 cells (Fig. 2E). These results suggest that PTP1B is capable of binding adapted to HCV infection in vitro (49). Remarkably, pretreatment to IFNAR1 and dephosphorylating this receptor. of these cells with PTP1B inhibitor 7-CBNP greatly decreased the Knockdown of PTP1B caused a noticeable increase in IFN- doses of IFN-α required to suppress HCV replication assessed by α–induced Tyr phosphorylation of IFNAR1 yet did not affect Ser- levels of HCV Core antigen (Fig. 4D). We further tested the role 535 phosphorylation or ubiquitination of this receptor (Fig. 3A), of PTP1B in defenses against VSV in MEFs and human 2fTGH indicating that PTP1B regulates endocytosis of IFNAR1 down- cells. VSV replication and its inhibition by IFN1 was comparable stream of its ubiquitination. Whereas turnover of IFNAR1 was in MEFs from either wild-type or PTP1B-null mice; furthermore, stimulated by both IFN-α and IL-1β, PTP1B knockdown notice- the effect of IFN1 was not augmented by PTP1B inhibitor 7- F ably attenuated the extent of this stimulation only in IFN- CBNP in wild-type MEFs (Fig. S2 ). Whereas VSV titer reached α– B fi 1,925,000 × 103 in IFNAR1 knockout MEFs reconstituted with chi- treated cells (Fig. 3 ), further suggesting that PTP1B speci - – cally regulates the ligand-inducible pathway of IFNAR1 degra- meric mouse human IFNAR1, and IFN1 alone decreased this titer to 445 × 103, a combined pretreatment with 7-CBNP and dation in human cells. Given that, of all known species, murine × 3 C IFN1 further decreased this titer to 45 10 . These results in- IFNAR1 does not contain Y466 or its analog (Fig. 3 )anddoes dicate that PTP1B may limit the antiviral effects mediated by not rely on this motif for IFNAR1 down-regulation (13), we fur- human IFNAR1. Indeed, knockdown of PTP1B in human 2fTGH ther used mouse cells as a control. Importantly, the extent of α D robustly augmented the ability of human IFN- to decrease in the IFN1-stimulated degradation (Fig. 3 ) or internalization (Fig. replication of VSV in a dose-dependent manner (Fig. 4E). Fur- S1F) of endogenous IFNAR1 was comparable between mouse thermore, treatment of 2fTGH with three diverse pharmacologic embryonic fibroblasts (MEFs) from wild-type mice and those from inhibitors of PTP1B dramatically sensitized VSV to replication animals lacking Ptpn1, the gene encoding PTP1B. This result suppression even by low doses of IFN-α (Fig. 4F). indicates that PTP1B plays no apparent role in regulating endo- In the absence of relevant mouse in vivo models, we carried out cytosis and stability of mouse IFNAR1 in mouse cells. a pilot clinical study on cats affected by chronic caudal stomatitis,

19228 | www.pnas.org/cgi/doi/10.1073/pnas.1211491109 Carbone et al. Downloaded by guest on September 28, 2021 Fig. 3. PTP1B regulates Tyr phosphorylation and the stability of IFNAR1 in human cells. (A) Endogenous IFNAR1 immunopuriﬁed from 293T cells that received indicated shRNA and were treated with IFN-α or IL-1β (as indicated) was analyzed by immunoblotting using the indicated antibodies. (B) Degradation of endogenous IFNAR1 in 293T cells transfected and treated as indicated was analyzed by immunoprecipitation–immunoblotting. Levels of actin in samples were also analyzed. (C) Primary sequence alignment of proximal fragments of the intracellular domains of IFNAR1 proteins from indicated species. Tyr-based endocytic motifs are depicted in bold. (D) MEFs from animals with indicated genotype were treated with PBS or murine IFN-β and CHX as indicated. Levels of murine IFNAR1 and actin were analyzed. (E)In- ternalization rate of chimeric mouse–human IFNAR1 proteins (chWT or chYF) coexpressed with PTP1B as indicated in MEFs from IFNAR1 knockout mice. (F) Turnover rate of FLAG-tagged mouse (mR1) or chimeric mouse–human IFNAR1 (chWT or chYF) expressed in WT or PTP1B-null MEFs treated with mIFN for indicated times.

a condition that is etiologically linked to feline calicivirus infection PTP1B-dependent acceleration of IFNAR1 endocytosis may (50) and could be alleviated by injections of feline IFN-ω (51). Pu- protect tissues from immunopathologic effects of IFN1. tative feline IFNAR1 sequence harbors a Tyr-based motif (Fig. 3C), PTP1B controls endocytosis of IFNAR1 downstream of its and 7-CBNP robustly augments STAT1 phosphorylation in response ubiquitination yet upstream of AP2 recruitment (Figs. 1 and 2). BIOCHEMISTRY to IFN1 in feline T cells (Fig. S3A). Accordingly, 2 wk after patients Given that PTP1B interacts with IFNAR1 and can dephosphor- receivedasinglesubmucosalinjection of 7-CBNP, a notable de- ylate this receptor (Fig. 2), a direct removal of phosphate from crease in inflammation score was observed in all five cats enrolled in Y466 and ensuing acceleration of IFNAR1 endocytosis would this study (Fig. S3 B and C). That included a marked reduction in be the simplest explanation for PTP1B-dependent regulation erythema, edema, and proliferative inflamed tissue volumes (Fig. of IFNAR1 stability and function (Fig. 4H). Although additional 4G). Together, these data strongly suggest that inhibition of PTP1B mechanisms such as stimulation of postinternalization sorting augments IFN1-elicited signaling and antiviral defenses. [proposed for the epidermal growth factor receptor (56)] or general interference with JAK–STAT phosphorylation (44, 57, 58) should Discussion not be ruled out, a direct involvement of Y466-driven endocytosis is evident from our studies. Indeed, mouse receptor lacks a Tyr-based Tyr-based linear endocytic motifs are known to serve as recogni- C tion sites for the AP2 complex (31). Whereas the role of the Y466- motif (Fig. 3 ), and PTP1B knockout mice display no abnormali- based motif in the internalization of IFNAR1 has been docu- ties in the IFN1 pathway (59, 60), except for a modestly augmented mented (12), the current paradigm predicted that the catalytic STAT1/3 phosphorylation in MEFs attributed regulation of JAK2 and TYK2 (44). Accordingly, whereas interaction of IFNAR1 with function of JAKs that determine the phosphorylation status of the TYK2 masks Y466 in unstimulated human cells to prevent basal Tyr residues within IFNAR1 [including Y466 (32)] might add down-regulation of this receptor (13–15, 61), mouse cells lacking another level of complexity to the regulation of IFNAR1 endocy- TYK2 display normal levels of IFNAR1 (16, 17). Furthermore, tosis and stability. Phosphorylation of Y466 is expected to reduce fi replacement of Tyr residues within murine IFNAR1 does not affect the af nity of AP2 components for the Tyr-based endocytic motif its signaling (62). Collectively, these results and our current data within IFNAR1 as it was described for CTLA4 (52–54). Indeed, β— clearly demonstrating the dependence of PTP1B-mediated effects current data reveal that IL-1 an inducer of IFNAR1 ubiquiti- on the integrity of Y466 in human and chimeric receptors strongly nation that does not activate JAK and stimulate IFNAR1 Tyr — fi suggest that dephosphorylation of Y466 represents a major mode phosphorylation is a more ef cient activator of IFNAR1 endo- of regulation. Indeed, a robust increase of antiviral effects of IFN1 α cytosis and degradation than IFN- (Fig. 1). Furthermore, given in vitro upon PTP1B inhibition seen in cells expressing human or α that IFN- still stimulates IFNAR1 endocytosis, we postulated that chimeric IFNAR1 but not mutant lacking Y466 or mouse receptor phosphorylation of Y466 might be reversed by a putative PTP to (Fig. 4 and Fig. S2E). enable IFNAR1 endocytosis and degradation (Fig. 4H). A genetic The latter data are of medical significance because they suggest screen and subsequent genetic, pharmacologic, and biochemical potential use of PTP1B inhibitors as antiviral drugs. Because we analyses revealed PTP1B to be a key determinant of ligand-de- could not use mice for in vivo studies, the proof-of-principle clinical pendent IFNAR1 endocytosis (Figs. 1 and 2) and degradation (Fig. experiments were carried out in a small group of feline patients 3) and an important regulator of IFN1 signaling and antiviral de- suffering from IFN-sensitive stomatitis (Fig. 4G and Fig. S3 B fense (Fig. 4). Whereas IFN1 protects from viruses, it is known to and C). Whereas the mechanism of beneficial effect of PTP1B mediate additional tissue damage in the context of many nonviral inhibitor seen in these cats is likely to be complex, these data infections and autoimmune syndromes (55). It is plausible that together with in vitro results provide a strong rationale for PTP1B

Carbone et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19229 Downloaded by guest on September 28, 2021 Fig. 4. PTP1B regulates the extent of IFN1 signaling and antiviral defenses. (A) Relative activity of ISRE– luciferase reporter expressed in MEFs that received human IFNAR2 (R2) indicated types of human IFNAR1 and PTP1B where indicated. The cells were left un- treated (white bars) or treated with mouse IFN-β (1,000 U/mL; black bars) or human IFN-α (1,000 U/mL; gray bars) for 5 h and harvested 40 h later. Normal- ized (per renilla lucifere activity) values from three independent experiments are presented. *P < 0.05 compared with all other groups. (B)ISRE–luciferase activity in human 2fTGH cells that received indicated shRNA and were treated or not with human IFN-α (1,000 U/mL for 30 min) and harvested 24 h thereafter was depicted as in A.(C)ISRE–luciferase activity in MEFs from IFNAR1 knockout mice reconstituted with chimeric IFNAR1 proteins (WT or YF, with or without PTP1B) and treated with mouse IFN-β where indicated. (D) Immunoblotting analysis of lysates from in Huh7.5 cells (pretreated with either PBS or 7-CBNP and subsequently treated with the indicated doses of IFN-α before being infected with HCV) was carried out using the anti-Core antigen antibody. Levels of β-actin are included as a loading control. (E)2fTGH cells that received either shControl (blue line) or shRNA against PTP1B (red line) were pretreated with the indicated doses of IFN-α for 2 h before being exposed to VSV [multiplicity of infection (MOI) 0.1 for 8 h]. The VSV viral titers in cell supernatants col- lected 16 h later were analyzed and depicted as an average of five independent experiments (each in triplicate correct). (F) 2fTGH cells that received either vehicle (DMSO or PBS; black) or PTP1B inhibitors (CinnGel2Me, 10 μM, blue; SSG, 10 μg/mL, red; or 7- CBNP, 150 μM, green) and the indicated doses of IFN-α for 2 h before being exposed to VSV (MOI 0.1 for 8 h). VSV titer was assessed as in E.(G) Thera- peutic effects of PTP1B inhibitor in feline patients with chronic viral stomatitis that failed standard anti-inflammatory treatment. Photos of selected lesions (white border area) pre- and postsubmucosal injection of 7-CBNP into the lesions are shown. (H) Scheme that summarizes signaling pathways that were established (30) and uncovered here. PTP1B counteracts JAK-induced phosphorylation of Y466- based endocytic motif whose exposure is required for efficient endocytosis upon IFNAR1 ubiquitination.

inhibition for augmenting therapeutic effects of either endoge- a gift from R. Harty, University of Pennsylvania, Philadelphia) as described (66). nous or exogenous IFN1. This approach may optimize IFN1- Replication of HCV in Huh7.5 cells was monitored by immunoblot analysis of based therapies in patients with viral infections (1–5), multiple HCV Core Antigen. Antibodies that recognize endogenous IFNAR1 (67) and sclerosis (63), and cancer (64). Future preclinical studies of IFNAR1 phosphorylated on Ser-535 (18) as well as assays for immunoprecipi- PTP1B inhibitors using non-mouse-based in vivo models are tations, immunoblotting, and assessment of the kinetics of IFNAR1 degrada- warranted. tion were described (13, 19, 68). IFNAR1 internalization assays were carried out by using a high-throughput fluorescence-based method as described (12). Methods The protein phosphatase inhibitors CinnGel2Me (Biomol International), Methods are outlined in detail in SI Methods. sodium stibogluconate (Santa Cruz Biotechnology), and CD45 inhibitor (Santa The siRNA oligonucleotides to knock down protein phosphatases or AP2 Cruz) were purchased. The synthesis and properties of the ZYZ inhibitor were from Qiagen. Human embryo kidney 293T cells, 293 cells that stably (Compound II) were described (41). The 7-CBNP was synthesized and purified express the human IL-1 receptor (293 Il-1RI; provided by J. Ninomiya-Tsuji, at Best West Laboratories as described (42) with the modification that used North Carolina State University, Raleigh, NC), MEFs of diverse genotypes, CuI/NaI/amine catalytic reaction condition, which enabled us to increase the 2fTGH cells (from G. Stark, Cleveland Clinic Foundation, Cleveland), and yield of intermediate 6-bromo-7-iodo-naphthonitrile. Huh7.5 (provided by C. Rice, Rockefeller University, New York) were grown in Dulbecco’smodified Eagle medium containing 10% (vol/vol) heat-inactivated ACKNOWLEDGMENTS. We thank J. Chernoff, R. Harty, C. Horvath, J. Krolewski, FBS and penicillin/streptomycin. Constructs for the expression of IFNAR2 and A. Mexas, B. Neel, J. Chernoff, J. Ninomiya-Tsuji, C. Rice, and G. Stark for ISRE-driven luciferase reporter (65) were gifts from J. Krolewski (University of reagents. This work was supported by National Institutes of Health Grants California, Irvine, CA) and C. Horvath (Northwestern University, Evanstone, IL). CA092900 and CA142425 (to S.Y.F.) and by a Mari Lowe Pilot grant (to S.Y.F. The antiviral effects of IFN were determined by using VSV (Indiana serotype; and J.R.L.).

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