Regulated Proteolysis of the Ifnar2 Subunit of the Interferon-Alpha Receptor

Regulated proteolysis of the IFNaR2 subunit of the interferon-alpha receptor

Abu ZM Saleh1, Aaron T Fang1, Allison E Arch1, Divas Neupane2, Ashraf El Fiky2 and John J Krolewski*1,2,3

1Department of Pathology, College of Medicine, University of California, Irvine, CA 92697, USA; 2Department of Biological Chemistry, College of Medicine, University of California, Irvine, CA 92697, USA; 3Chao Family Comprehensive Cancer Center, College of Medicine, University of California, Irvine, CA 92697, USA

The type I interferons (IFNs) bind surface receptors, modulate gene transcription (Levy and Darnell, 2002). induce JAK kinases and activate STAT transcription The JAKs bind constitutively to receptor subunits, are factors to stimulate the transcription of genes downstream activated when ligand binding dimerizes the receptor of IFN-stimulated response elements (ISREs). In this subunits and sequentially phosphorylate multiple com- study, we demonstrate that IFNaR2, a subunit of the type ponents of the signaling cascade. In the case of the type I I IFN receptor, is proteolytically cleaved in a regulated IFNs, including IFN-a and -b, ligand binding to the manner. Immunoblotting shows that multi-step cleavage IFNaR1 and IFNaR2 receptor subunits juxtaposes and occurs in response to phorbol ester (PMA) and IFN-a, activates Tyk2 and Jak1, the associated JAK kinases generating both a transmembrane ‘stub’ and the intracel- (Colamonici et al., 1994; Domanski et al., 1995; Yan lular domain (ICD), similar to Notch proteolysis. Isolated et al., 1996b). The JAKs become phosphorylated, and in membrane fractions process IFNaR2 to release the ICD. turn phosphorylate a critical tyrosine on IFNaR1, which A chimeric receptor construct is utilized to show that functions as a docking site for the Stat2 SH2 domain cleavage requires the presenilins and occurs in response to (Yan et al., 1996a; Li et al., 1997). Subsequently, Stat2 epidermal growth factor and protein kinase C-d over- and then Stat1 are phosphorylated, heterodimerize and expression, as well as PMA and type I IFNs. Fluorescence translocate to the nucleus to stimulate transcription. microscopy demonstrates that a green ﬂuorescent protein– Regulated intramembrane proteolysis (RIP) has ICD fusion localizes predominantly to the nucleus. A emerged as a mechanistically simple and evolutionarily fusion between the ICD and the Gal4 DNA-binding conserved mechanism for relaying a signal from a domain represses transcription, in a histone deacetylase- membrane surface to the nucleus (Brown et al., 2000). dependent manner, of a Gal4 upstream activating An initial, regulated cleavage within a juxtamembrane sequence-regulated reporter, while overexpression of the (JM) region of the extracellular domain releases the ICD alone represses transcription of a reporter linked to extracellular domain. The residual membrane-bound an ISRE. Proteolytic cleavage events may facilitate ‘stub’ is further processed by an intramembrane (IM) receptor turnover or, more likely, function as a mechanism protease, releasing the intracellular domain (ICD), for signaling similar to that employed by Notch and the which translocates to the nucleus and modulates gene Alzheimer’s precursor protein. transcription or other biological activities (Weihofen Oncogene (2004) 23, 7076–7086. doi:10.1038/sj.onc.1207955 and Martoglio, 2003). For many of the known Published online 2 August 2004 eukaryotic RIP substrates, intramembrane proteolysis is catalysed by g-secretase, a multi-protein complex Keywords: interferon; intramembrane protease; which includes one or both of the presenilin proteases presenilin; protein kinase C (Capell et al., 1998; Yu et al., 1998; Li et al., 2000; Esler et al., 2002; Takasugi et al., 2002) and a set of associated proteins (Edbauer et al., 2003; Kimberly et al., 2003; Takasugi et al., 2003). Some membrane proteins, such as Introduction sterol regulatory element binding protein 2 (SREBP-2) and Notch, appear to signal primarily via the RIP Receptors for the large family of helical cytokines, mechanism. The ICD can function to modulate gene which include the interferons (IFNs), utilize JAK transcription via direct DNA binding (SREBP-2) or by tyrosine kinases and STAT transcription factors to interacting with DNA-bound protein complexes (Notch). The receptor type tyrosine kinases HER4 (Ni et al., 2001) and the M-CSF-1 receptor (Wilhelmsen and *Correspondence: JJ Krolewski, Department of Pathology, University van der Geer, 2004) can be cleaved in a regulated of California, Irvine, Medical Sciences I D450, Irvine, CA 92697-4800, USA; E-mail: [email protected] manner resembling RIP, suggesting that these molecules Received 9 March 2004; revised 10 June 2004; accepted 15 June 2004; might signal via canonical tyrosine kinase-driven mecha- published online 2 August 2004 nisms as well as RIP-mediated mechanisms. Thus, we IFNaR2 receptor proteolysis AZM Saleh et al 7077 sought to determine whether a member of the helical plasma membrane and cytosolic fractions and immuno- cytokine receptor family might be similarly processed. blotted with an anti-HA antibody. The membrane Our results demonstrate that IFNaR2 can be cleaved in fraction from cells expressing the full-length receptor response to IFN-a and other stimuli, including activated contained both the 97 kDa intact receptor and a 58 kDa protein kinase C-d (PKCd), and that the IFNaR2 ICD is cleavage product (Figure 1c, lane 4). Reprobing with an capable of nuclear translocation and transcriptional anti-EF2 antibody (Figure 1c, lower panel) indicates modulation. that the membrane fraction was essentially free of cytoplasm (EF2 is cytoplasmic). To determine the

Results

Proteolytic cleavage of IFNaR2 To investigate IFNaR2 processing, we exploited the observation that phorbol ester can activate the metalloprotease TACE, which is known to mediate the initial regulated JM cleavage event in the RIP signaling of HER4 (Rio et al., 2000), as well as other transmembrane proteins (Okamoto et al., 1999; May et al., 2002). U5A cells, which do not express the IFNaR2 subunit of the IFN-a receptor (Lutfalla et al., 1995), were stably transfected with an IFNaR2-hemagluttinin (HA) construct containing an HA epitope tag at the extreme carboxyl terminus (Nguyen et al., 2002). A stable transfectant expressing IFNaR2 was treated with phorbol ester for varying times and extracts were immunoblotted with an anti-HA antibody. A number of novel bands migrating at approximately 58, 40, 34 and 27 kDa (faint) were detected (Figure 1a). To characterize the PMA-generated IFNaR2 fragments, we transiently expressed the three constructs shown in Figure 1b: the full-length IFNaR2-HA construct used in Figure 1a (FL), and two variants in which large portions of the extracellular domain have been deleted (E1 and E2). These HEK293T cell transfectants were treated with PMA, separated into

Figure 1 Proteolytic cleavage of IFNaR2. (a) PMA-dependent cleavage of IFNaR2. U5A cells stably expressing an IFNaR2 gene containing a carboxyl-terminal HA tag were treated with 100 ng PMA/ml for the indicated time (min) and lysed with 1% NP40. The lysate was centrifuged and the supernatant was immunoblotted and probed with an anti-HA antibody. The approximate sizes of various fragments are indicated. Probing of nontransfected lysates conﬁrmed that all of the observed bands were IFNaR2-speciﬁc (data not shown). (b) IFNaR2 constructs used in (c). The full- length (FL) construct and two internally deleted variants (E1 and E2) are shown. The amino terminus is at the top, followed by the signal sequence, corresponding to residues 1–28 (gray rectangle). The black rectangle in the middle represents the transmembrane domain (residues 244–264) and gray oval represents the carboxyl- terminal HA tag. In E1, residues 45–219 were deleted and a FLAG tag inserted. In E2, residues 33–232 were deleted. (c) Subcellular localization of IFNaR2 cleavage products. HEK293T cells were transfected with plasmids encoding the IFNaR2 constructs depicted in (b). After 2 days, proteins were extracted as in (a) (M þ C), or from separate cell membrane (M) or cytosolic (C) fractions. (d) IFNaR2 cleavage in isolated membranes. U5A cells stably expressing IFNaR2-HA were treated with PMA for 90 min, membrane preparations were prepared as detailed in Materials and methods and subsequently incubated for 2 h at the indicated temperature. Following in vitro incubation, the membrane preparations were centrifuged and the pellet (Mem) and supernatant (Sup) fractions immunoblotted and probed with anti-HA antibody

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7078 approximate location of the cleavage site, we compared the migration of the FL, E1 and E2 constructs. The 58 kDa band generated from FL IFNaR2 (lane 4) co- migrates with the E2 deletion construct (lane 6), suggesting that IFNaR2 has been cleaved near residue 233. Construct E1 encodes a protein that is 33 amino acids longer than E2 and clearly migrates more slowly (lane 5). Thus, the initial (JM) cleavage event which removes the bulk of the ecto-domain occurs in the vicinity of residue 233, which is about 11 amino acids proximal to the amino terminus of the predicted IFNaR2 transmembrane domain. Examination of the cytosolic fraction indicates that a 34 kDa protein is generated from all the three IFNaR2 constructs (lanes 7–9). The B40 kDa band observed in Figure 1a is only weakly detected in this experiment. The presence of the 34 kDa bands in the cytosol of the E1 and E2 constructs is consistent with the idea that constructs which are engineered to resemble membrane-bound ‘stubs’ are constitutively processed by an IM protease to release the corresponding ICD (Struhl and Greenwald, 2001; LaVoie and Selkoe, 2003). A 27 kDa fragment is also detected in the cytosol of the FL construct (lane 7). In nuclear fractions from cells transfected with the FL and E2 constructs, both the 27 and 34 kDa bands were observed, with the 27 kDa more prominent (AE and JJK, unpublished results). To further investigate the IM cleavage of IFNaR2, cells employed in Figure 1a were treated with PMA and the partially purified plasma membrane preparations were incubated at either 4 or 371. These membrane fractions are expected to contain full-length IFNaR2, the 55 kDa ‘stub’ and the putative processing proteases. Following incubation, membranes were pelleted and the companion supernatants, containing soluble proteins released by intramembrane proteolysis, were collected. Proteins from the pelletted membranes (Mem) as well as the supernatant (Sup) were immunoblotted with anti- Figure 2 Cleavage of IFNaR2 in response to IFN-a.(a) Cells and HA antibody. In the supernatant fraction, we observed protein lysates were the same as used in Figure 1a, except that cells that a 34 kDa protein was generated more efficiently at were treated with 3000 U IFN-a2/ml for the indicated time (min). Lysate corresponding to either 2 mgor20mg of protein was 371, relative to 41 (Figure 1d, lane 8), consistent with immunoblotted with an anti-HA antibody. The blot containing proteolytic cleavage of the IFNaR2 transmembrane 20 mg of protein was exposed longer to visualize the smaller domain by protease(s) residing in the plasma membrane. fragments. The blot containing 2 mg of protein was stripped and re- As in Figure 1c (lane 7), a less abundant 27 kDa protein probed with an anti-EF2 antibody. (b, c) IFN-a-induced cleavage 1 of IFNaR2 in U5A transfectants expressing lower levels of also increases following incubation at 37 . In addition, IFNaR2-HA protein. Similar to (a), except that the cell line used there is a decrease in the amount of full-length IFNaR2 in (b) expresses IFNaR2-HA at about 10% the level of the cell line (compare lanes 1 and 2). There is a small amount of the in (a), while the line used in (c) expresses about 3% as much 58 kDa protein that corresponds to the JM cleavage protein, based on comparative immunoblots (data not shown). In product (lanes 5–6). (b), lysate corresponding to either 10 mg (lanes 1–3) or 50 mg (lanes 4–6) of protein was immunoblotted. In (c), lysate corresponding to To determine if a physiologic ligand for the type I 50 mg of protein was immunoblotted. Two exposures of the same IFN receptor could induce IFNaR2 cleavage, we treated anti-HA immunoblot are shown the U5A-IFNaR2-HA cells used in Figure 1a with IFN- a2 for up to 2 h and immunoblotted the extracts using an anti-HA antibody (Figure 2a). The level of the full- selected two that expressed considerably lower levels of length IFNaR2 protein declined in a time-dependent IFNaR2-HA. Specifically, the lines examined in Figures fashion, indicating that IFN-a activated the proteolytic 2b and c expressed IFNaR2-HA at about 10 and 3% of process. A second immunoblot of the same lysates, the level of expression observed in the line used in containing 10-fold more protein, demonstrated an IFN- Figure 2a (data not shown). Both of these lines also a-dependent increase in the various cleavage products, display IFN-a-dependent reduction of full-length IF- including the 58, 40 and 34 kDa species. We generated NaR2 as well as production of the 35 kDa cleavage additional U5A-IFNaR2-HA-transfected cell lines, and product (Figure 2b, c).

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7079 Presenilins effect IFNaR2 cleavage type PS1 show a significant increase in luciferase activity compared to cells receiving vector DNA. In contrast, To further characterize IFNaR2 cleavage, we con- cells expressing a catalytically inactive version of PS1, structed a ‘cleavage reporter’ in which most of the in which a critical aspartate residue has been converted ICD of IFNaR2 was replaced by a Gal4DBD–VP16 to alanine, reveal reduced cleavage activity. Cells co- transactivation domain (TAD) fusion sequence expressing catalytically inactive versions of both PS1 (Figure 3a) (Karlstrom et al., 2002; May et al., 2002). and PS2 do not cleave the IFNaR2 reporter construct at Intramembrane cleavage of this protein (I2-ECD-GV) all, relative to control cells transfected with vector releases the Gal4–VP16 fusion protein fragment, which DNA. Some individuals afflicted with familial Alzhei- in turn translocates to the nucleus and stimulates mer’s disease (FAD) have germline mutations in the PS1 transcription of a co-transfected luciferase gene under gene (Cruts and Van Broeckhoven, 1998), which appear the control of an element that binds the Gal4DBD. to alter the pattern of intramembrane cleavage of the Figure 3b demonstrates that one or both presenilin gene Alzheimer’s precursor protein (APP). When the plasmid products mediate proteolytic cleavage of I2-ECD-GV. encoding I2-ECD-GV was transfected into CHO cells Specifically, a set of CHO cell derivatives (Kimberly expressing the M146V FAD mutant of PS1, cleavage et al., 2000) stably expressing either wild-type presenilin was reduced, relative to cells expressing wild-type PS1. 1 (PS1) or various mutant forms of PS1 or PS2 were transiently transfected with pMT-I2-ECD-GV (or vector) and pG4-thymidine kinase (TK)luc, which encodes Multiple effectors of IFNaR2 cleavage a luciferase gene under the control of a Gal4 upstream To confirm and extend the cleavage data in Figures 1 activating sequence (UAS). CHO cells expressing wild- and 2, we again employed the chimeric I2-ECD-GV construct. Figure 4 indicates that this form of IFNaR2 is cleaved in untreated cells, as observed in Figure 3. Treatment with IFN-a2 enhances the cleavage of I2- ECD-GV about twofold, while IFN-b, which also binds the IFNaR1–IFNaR2 receptor complex, induces cleavage to a similar extent. As expected, PMA enhances IFNaR2 cleavage, but it does not synergize with IFN-a2 (Figure 4). Finally, epidermal growth factor (EGF) is also capable of stimulating cleavage of the I2-ECD-GV protein (Figure 4).

PKCd regulates IFNaR2 cleavage To investigate the possibility that PKCd, which is induced by IFN-a (Uddin et al., 2002), mediates the

Figure 3 Presenilin 1 is required for intramembrane proteolysis of IFNaR2. (a) Structure of cleavable reporter construct (I2-ECD- GV). Residues 1–299 of IFNaR2, spanning the extracellular domain (ECD), the transmembrane domain (black rectangle) and the ﬁrst 32 amino acids of the ICD were fused to the 147-residue DNA-binding domain of the yeast Gal4 protein (Gal4DBD) and the 78-residue transcriptional activation domain of VP16 (VP16TAD) to create pMT-I2-ECD-GV. (b) Cleavage of IFNaR2 is mediated by PS1 and PS2. CHO cell lines stably expressing wild- type presenilin 1 (PS1wt); a mutant form of PS1 found in individuals with FAD, in which the methionine at position 146 has been replaced by valine; a mutant form of PS1 in which aspartic acid 257 has been converted to alanine (PS1D257A); or both mutant Figure 4 Type I IFNs, PMA and EGF stimulate intramembrane PS1D257A and an analogous mutant of PS2 in which aspartic acid cleavage of IFNaR2. HEK293T cells were transfected with pMT- 366 has been converted to alanine (PS1D257A þ PS2D366A), were I2-ECD-GV ( þ ) or pMT2T (À) plasmids, as well as pG4-TKluc transiently transfected with pMT-I2-ECD-GV ( þ ) or pMT2T (À) and pRL-TK. After 1 day, cells were left untreated (None), or plasmids as indicated, as well as pG4-TKluc and pRL-TK treated with 3000 U/ml of either IFN-a or IFN-b, 100 ng/ml PMA, (transfection efﬁciency control). After 2 days, luciferase activities both PMA and IFN-a, or 20 ng/ml EGF. After 24 h, luciferase were measured and normalized as described in Materials and activities were measured and normalized as described in Materials methods and methods

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7080 cleavage of IFNaR2, we transfected HEK293T cells with either vector DNA or plasmids encoding the wild- type or kinase-deficient forms of murine PKCd,in addition to the same cleavable reporter (pMT-I2-ECD- GV) and luciferase constructs (pG4-TKluc) employed in Figures 3 and 4. Expression of wild-type PKCd stimulated cleavage, relative to either the kinase- deficient version or vector (Figure 5). A similar experiment demonstrated that the pan-PKC inhibitor BIM-I (Martiny-Baron et al., 1993) inhibited PKCd-mediated cleavage (Figure 6a). Rottlerin, which is a PKC-d- specific inhibitor at low concentrations (Gschwendt et al., 1994), was also able to inhibit cleavage (Figure 6b). The inhibitor Go¨ 6976, which targets classical PKC proteins (Martiny-Baron et al., 1993) (PKCa, bI, bII and g), had no effect (Figure 6c). The g-secretase inhibitor H5106 blocks PKCd-mediated cleavage of I2-ECD-GV, again indicating that the molecular mechanism involves intramembrane proteolysis (Figure 7).

Nuclear localization of the IFNaR2 ICD To investigate the ability of the IFNaR2 ICD to translocate to the nucleus, U2OS cells were transfected with a construct encoding a green fluorescent protein (GFP)–ICD fusion (GFP–I2–ICD). A GFP–HER2– ICD construct, known to localize primarily to the cytoplasm (Ni et al., 2001), was employed in a parallel, control transfection. Figure 8 shows that the GFP–I2– ICD was predominantly seen in the nucleus. When unfused GFP was expressed in the same cells, green fluorescence was detected throughout the cell, albeit somewhat more intensely in the nucleus (data not shown). However, the ratio of nuclear to cytoplasmic fluorescence was substantially greater in the cells transfected with GFP–I2–ICD, relative to those expressing unfused GFP. Interestingly, we observed relatively

Figure 6 Effects of PKC inhibitors on PKC-stimulated intramembrane cleavage of IFNaR2. Similar to Figure 5, except that cells received either plasmids encoding wild-type PKC ( þ ) or vector (À). The indicated inhibitors were added to the media at the concentrations shown for the ﬁnal 48 h prior to termination of the cultures

Figure 5 Protein kinase C-d stimulates intramembrane cleavage of IFNaR2. HEK293T cells were transfected with pMT-I2-ECD-GV, few green fluorescent cells in the culture transfected pG4-TKluc and pRL-TK plasmids, as well as either vector DNA with GFP–I2–ICD compared to the GFP–HER2–ICD (vector), a plasmid encoding a kinase-deficient version of the murine PKCd gene (PKC-KD) or a wild-type version (PKC-WT). control (data not shown). This is consistent with the After 2 days, luciferase activities were measured and normalized as finding that ICDs are relatively labile (Brown et al., described in Materials and methods 2000).

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7081 The IFNaR2 ICD represses gene transcription Given that IFNaR2 can be proteolysed to generate an ICD-sized fragment, and that this fragment is capable of nuclear translocation, we sought to determine if the putative IFNaR2 ICD could modulate gene transcription. Initially, we tested the transcriptional effects of the IFNaR2 ICD by fusing this fragment to the Gal4DBD. The resulting construct (Gal4–I2–ICD) was transfected, along with a Gal4 UAS-regulated luciferase reporter gene, into U2OS cells and reporter gene activity was assayed. Surprisingly, Gal4–I2–ICD repressed reporter gene activity, relative to cells transfected with the Gal4DBD construct alone (Figure 9a). All other previously characterized ICDs produced by RIP have been shown to enhance transcription (Ebinu and Yanker, 2002). The ability of the IFNaR2 ICD to Figure 7 Effect of g-secretase inhibitor on PKC-mediated cleavage of IFNaR2. Similar to Figure 6, except that the inhibitor repress gene transcription is dependent on the amount H5106 was added at the indicated concentration

Figure 9 The IFNaR2 ICD represses reporter gene transcription. Figure 8 Nuclear localization of a GFP IFNaR2 ICD fusion (a) A Gal4DBD IFNaR2 ICD fusion represses reporter gene protein. U2OS cells were transfected as described in Materials and transcription. U2OS cells were transfected with a plasmid encoding methods, fixed, counterstained with a nuclear dye and viewed the Gal4DBD alone (Gal4) or a plasmid that encodes the IFNaR2 under a fluorescence microscope. The two left-most columns ICD fused to the Gal4DBD (Gal4–I2–ICD), pG4-TKluc and a contain images of representative cells transfected with a GFP– plasmid encoding a constitutive b-galactosidase gene (transfection HER2–ICD. Images of green fluorescence are shown in the first efficiency control). Lysates were assayed for luciferase and b- column and corresponding images of blue fluorescence (nuclear galactosidase activities and normalized luciferase activity is plotted. staining) are shown in the second column. The two right-most (b) The IFNaR2 ICD represses gene transcription in a dose- columns show equivalent images from representative cells trans- dependent manner. Similar to (a), except that varying amounts of fected with a GFP–IFNaR2 ICD construct the Gal4–I2–ICD-encoding plasmid were used

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7082

Figure 11 The IFNaR2 ICD represses transcription of an ISRE reporter. U5A cells were transfected as described in Materials and methods and subsequently assayed for luciferase activities. Cultures were transfected either with constructs encoding the IFNaR2 ICD (left) or the corresponding vector (right). Each ﬁreﬂy luciferase value was normalized by dividing by the corresponding Renilla luciferase value. The normalized luciferase value obtained from cells transfected with the ISRE-luc construct was divided by the normalized luciferase value for cells receiving pGL3 vector

To determine if the IFNaR2 ICD can modulate transcription from the ISRE, U5A cells were transiently transfected, pairwise, with either a plasmid expressing an open reading frame corresponding to the ICD or the corresponding vector, and either an ISRE-linked luciferase reporter or the corresponding vector (see Materi- als and methods). In this set of reporter assays, the transcriptional effect of the ICD on an ISRE linked to a minimal TK promoter was normalized to its effect on the minimal promoter alone. Thus, Figure 11 shows the effect of either an empty vector (left column) or the IFNaR2 ICD (right column) on an ISRE-luciferase reporter relative to the effect on the companion luciferase reporter plasmid containing only a minimal promoter. As was the case with the Gal4DBD–I2–ICD Figure 10 TSA inhibits IFNaR2 ICD-mediated transcriptional fusion, the isolated ICD repressed transcription from a repression. Similar to Figure 9a, except that TSA at the indicated reporter construct containing a response element linked concentration was added to cultures for the ﬁnal 24 h. (Inset) The to a minimal TK promoter. data from the three sets of transfected cells were re-plotted as percent repression, as a function of TSA concentration. TSA increased the transfection efﬁciency of the constitutively expressed b-galactosidase construct in a dose-dependent manner. Thus, the Discussion levels of normalized luciferase activity are higher in those cultures receiving TSA relative to the control cultures Intracellular signaling events, often depicted as linear cascades, are biochemically complex, involving crosstalk and feedback control as well as multiple mechanisms for of transfected plasmid DNA, suggesting a dependence initiation. RIP signaling is an ancient mechanism for on the level of ICD protein expression (Figure 9b). One relaying signals from the cell surface to the interior of mechanism for repressing gene transcription involves the cell. In the case of membrane proteins such as the recruitment of a histone deacetylase (HDAC). To SREBP-2, Notch and APP, intramembrane proteolysis test the possibility that one or more HDAC proteins appears to represent the only mechanism of signal mediate the observed repression, we repeated the initiation. In contrast, the recent discoveries that HER4 experiments shown in Figure 9a in the presence of and the M-CSF-1 receptor, two receptor-type tyrosine varying concentrations of trichostatin A (TSA), an kinases, undergo RIP in response to ligand binding HDAC inhibitor (Yoshida et al., 1990). Figure 10 suggests that some membrane proteins might signal via demonstrates that increasing concentrations of TSA intramembrane proteolysis of the ICD as well as, or in relieve the transcriptional repression produced by Gal4– lieu of, well-established mechanisms involving kinase I2–ICD, suggesting that one or more of the HDAC activation. We now provide additional evidence in proteins are involved in these inhibitory events. support of this possibility, by demonstrating that the

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7083 ICD of the IFNaR2 subunit of the type I IFN receptor induces multiple PKC isoforms as well as TACE, and can be cleaved in a regulated manner, can enter the EGF (Figure 4) also induce IFNaR2 cleavage. These nucleus and modulate gene transcription. observations suggest that if regulated proteolysis of First, immunoblotting studies demonstrate that IF- IFNaR2 does stimulate intracellular signaling, there is NaR2 can be proteolytically cleaved in the sequential ample opportunity for crosstalk with other signaling manner typical of RIP. Treatment of cells expressing mechanisms. IFNaR2 with PMA, which can activate TACE, a Importantly, two type I IFNs, IFN-a and IFN-b, can metalloprotease implicated in ecto-domain proteolysis also stimulate IFNaR2 cleavage, as indicated by both of multiple membrane proteins (Buxbaum et al., 1998; the immunoblotting studies (Figure 2) and the cleavable Rio et al., 2000; Black, 2002; Weskamp et al., 2004), reporter assay (Figure 4). We observed IFN-stimulated generates a membrane stub of IFNaR2 (Figure 1) as cleavage in transfected cells expressing significantly well as smaller fragments localized to the cytoplasm and different levels of the receptor subunit (Figure 2). nucleus. Co-migration with ecto-domain deletion deri- Although at this point we are uncertain how the levels vatives indicates that the larger (B58 kDa) cleavage of exogenous IFNaR2 in the various lines examined product is a membrane stub (Figure 1c). Based on compare to endogenous levels in typical cells, our data primary amino-acid sequence data, the E2 extracellular argue that IFN-mediated cleavage is not simply an domain deletion derivative (Figure 1c, lanes 3 and 6) artifact of overexpression. The kinetics of cleavage are should have a molecular mass of B36 kDa. The distinct from those observed for the tyrosine phosphor- apparent larger size of B58 kDa may be due to post- ylation of JAK-STAT components following IFN translational modification. Recent NMR structural treatment of the same cells. Specifically, tyrosine studies of IFNaR2 indicate that the region containing phosphorylation typically reaches a maximum within the cleavage site predicted by our data lies immediately the first half-hour following ligand treatment (Colamo- distal to a well-defined fibronectin-like domain of the nici et al., 1994), while we observed a continuing IFNaR2 ectodomain, within a short, stalk-like juxta- increase in proteolysis beyond 1 h (Figure 2). The membrane region (Chill et al., 2003). Similarly, the amount of the ICD generated in response to IFN TACE cleavage site in the growth hormone receptor treatment appears to be less than the extent of 97 kDa also resides in a structurally similar JM region cleavage. There are a number of possible explanations (Baumann and Frank, 2002). It appears that both stalk for this observation, including rapid translocation to the length and sequence are critical determinants of TACE nucleus and the fact that RIP substrates are relatively cleavage (Hinkle et al., 2004). Further experimentation labile. The full-length protein might also undergo is needed to determine whether TACE is indeed lysosomal degradation (Kumar et al., 2003). We also responsible for JM cleavage of IFNaR2, and to observed less of the 58 kDa stub in U5A cells relative to determine the actual cleavage site. HEK293T cells following treatment with PMA In addition, membrane-bound IFNaR2 can be (Figure 1), as well as in the absence of treatment (data processed in vitro, in a constitutive fashion, to yield not shown). This may be due to differences between soluble fragments (Figure 1d), consistent with our these cell lines in the relative efficiency or extent of suggestion that the responsible protease is in the plasma induction of one or both proteases involved in IFNaR2 membrane. These fragments (34 and 29 kDa) are processing. identical in size to those seen in the cytosol of PMA- PMA and IFN-a2 effects on cleavage are not additive treated cells (Figure 1c). If the intramembrane cleavage (Figure 4), suggesting that these agents act through occurs near the carboxyl terminus of the transmembrane similar, rather than complementary, signaling pathways. domain, the IFNaR2 ICD is predicted to have a mass of It has been reported that PKCd activity is induced by 27 kDa. Thus, as noted for the 58 kDa JM-cleavage IFN-a (Uddin et al., 2002). Consistent with this product, the 35 kDa fragment might be a post-transla- observation, we found that overexpression of PKCd tionally modified version of the ICD. Alternatively, was able to induce IFNaR2 cleavage (Figure 5) and that cleavage may occur at multiple positions within the this effect could be blocked by specific PKC inhibitors transmembrane domain, as has been observed for APP (Figure 6). The possible role of PKC in IFNaR2 RIP is (De Strooper et al., 1999; Okochi et al., 2002) as well as further strengthened by our finding that EGF, an other PS substrates (Xia and Wolfe, 2003). Protein activator of PKC (Rhee, 2001), can also stimulate sequencing of the cleavage products will define the site(s) cleavage of the IFNaR2 reporter (Figure 4). However, of cleavage. the molecular apparatus linking IFN-a and PKCd Our studies identified multiple stimuli that are capable activation has not been characterized and thus a of activating proteolysis of IFNaR2. Specifically, mechanistic role for PKCd in the regulated proteolysis cleavage occurs constitutively when cells are grown in of IFNaR2 remains uncertain and represents an area for mitogen-rich media (for example, Figure 2a, lane 1 and future investigation. the columns labeled ‘none’ in Figure 4). Such basal In addition to a sequential pattern of ligand-induced cleavage was also observed in serum-starved cells (data proteolysis, many RIP signaling pathways modulate not shown). Constitutive cleavage is frequently observed gene transcription. This requires obligatory transloca- for shed proteins and, in the case of TNFa, has been tion of the ICD to the nucleus, as suggested by our blocked by inhibiting p38 MAP kinase signaling (Fan observations on the GFP–ICD fusion in Figure 8. and Derynck, 1999). PMA (Figures 1 and 4), which Intramembrane cleavage could function to unmask a

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7084 latent nuclear localization sequence within the ICD; used according to the respective manufacturer’s directions. alternatively, an interacting protein might supply such a The anti-EF2 antibody was from K Nastiuk (University of signal. A fusion between the Gal4DBD and the IFNaR2 California, IRVINE). IFN-a2 was from M Brunda (Roche) ICD is, indeed, able to modulate the transcription of a and IFN-b1A was from JRosa (Biogen). The U2OS cell line Gal4UAS-driven reporter. Surprisingly, the transcrip- was obtained from ATCC, the CHO cell lines stably transfected with various wild-type and mutant presenilin genes tion of this reporter is repressed, in a dose-dependent (Kimberly et al., 2000) were from D Selkoe (Harvard manner, by the Gal4–I2–ICD fusion protein (Figure 9). University Medical School) and the HEK293T cell line was TSA, which can inhibit HDACs, was able to suppress from H Young (Columbia University, College of Physicians the transcriptional repression effect (Figure 10), suggest- and Surgeons). The IFNaR2-deficient U5A cell line, comple- ing that one or more HDAC proteins are involved in mented with a carboxyl-terminal HA-tagged IFNaR2 con- this process. Our preliminary experiments also indicate struct, has been described previously (Nguyen et al., 2002). that an isolated ICD might act similarly to repress Cells were grown in Dulbecco’s modified Eagle’s media transcription of a representative ISRE. How the ICD (Mediatech) plus 10% fetal calf serum (Atlanta Biologicals). binds the ISRE is not yet known. The reporter construct employed in these experiments contains functional Sp1 DNA constructs sites (Jones et al., 1985; Kubota et al., 2001). The Gal4– Plasmids encoding the following constructs have been pre- I2–ICD may recruit HDAC protein(s), which in turn viously described: a fusion between the GFP and the ICD of deacetylates histones at the transcription complex the human HER2 gene (pGFP–H2–ICD) (Ni et al., 2001), formed around one or both of these sites. from G Carpenter (Vanderbilt University); wild-type and The ICD proteins characterized thus far are quite kinase-deficient murine PKCd (Soh et al., 1999), from B variable in domain composition and function (Ebinu Weinstein (Columbia University, College of Physicians and and Yanker, 2002). One, SREBP, has both DNA- Surgeons); a firefly luciferase gene under the control of five binding domain (DBD) and TAD domains and thus copies of the Gal4UAS fused to 135 bp of the Herpes Simplex resembles a prototypical transcription factor. Others, virus TK promoter (pG5-TKluc) from JChan (University of exemplified by the APP ICD, appear to lack both DBD California, IRVINE) (Jones et al., 1985); and a constitutively and TAD domains and function either as adaptors or to expressed, TK promoter-driven Renilla luciferase gene (pRL- TK) (Promega). facilitate nuclear localization. Although we are uncer- A plasmid encoding a cleavable reporter derivative of tain what function(s) the IFNaR2 ICD itself provides, in IFNaR2 (pMT-I2-ECD-GV) was prepared by PCR amplify- separate experiments employing the Gal4DBD-ICD ing a cassette containing the yeast Gal4 DBD fused to the fusion (El Fiky, Arch and Krolweski, submitted), we VP16 TAD, incorporating NcoI and EcoR1 restriction sites at have determined that the transcriptional effects of the the 50 and 30 ends, respectively. The resulting fragment was IFNaR2 ICD are mediated by the TAD of Stat2, which sequenced and inserted into an endogenous IFNaR2 NcoI site has been shown previously to bind constitutively to the and a flanking EcoR1 site to create a fusion construct IFNaR2 ICD (Li et al., 1997; Nadeau et al., 1999; consisting of the complete extracellular and transmembrane Nguyen et al., 2002; Saleh et al., 2002). domains of IFNaR2, 32 amino acids of the ICD (extending to Although we favor the idea that proteolytic cleavage amino-acid position 299 of human IFNaR2) and a Gal4DBD– VP16TAD cassette, immediately followed by a stop codon. of IFNaR2 facilitates RIP signaling, there are other This fusion was transferred into the pMT2T eukaryotic possible biological reasons for these events. For expression vector. A fusion between GFP and the IFNaR2 instance, cleavage might be part of a general mechanism ICD was constructed by first amplifying a carboxyl-terminal for receptor protein turnover, with final degradation in fragment containing the IFNaR2 ICD, incorporating EcoR1 the nucleus. Alternatively, cleavage might be primarily restriction sites at each end. This fragment, which extends from employed as a mechanism for shedding the ectodomain, amino acid 266 to the carboxyl terminus (residue 515) and thereby generating a soluble receptor, which could bind corresponds to the region immediately distal to the predicted and neutralize extracellular type I IFNs and attenuate transmembrane domain, was cloned, sequenced, digested with antiviral activity. Thus, while we have documented some EcoR1 and ligated into the pEGFP-C3 vector (Invitrogen) to of the key molecular events indicative of RIP signaling – produce a GFP–I2–ICD fusion. There are 11 amino acids (YSDLELKLRIR) between the EGFP sequence and the regulated ectodomain cleavage, constitutive intramem- IFNaR2 ICD. To produce a fusion construct (pSG– brane cleavage, nuclear accumulation of, and transcrip- Gal4DBD–I2–ICD) encoding Gal4DBD-I2-ICD, the same tional modulation by, the ICD – further functional PCR product was digested with EcoR1, but in this case the studies will be required to determine whether the resulting overhang was filled using the Klenow polymerase IFNaR2 ICD mediates any of the effects of the type I fragment and ligated into the SmaI site of pSG424 (Sadowski IFNs. and Ptashne, 1989), which encodes the Gal4DBD (residues 1– 147). There are five amino acids (PELIR) between Gal4DBD codon 147 and the IFNaR2 ICD. Intact pSG242 was employed to express the Gal4DBD alone. Gal4UAS-regulated Materials and methods luciferase reporter gene activity was assayed using pG5-TKluc (Forman et al., 1995), which contains five Gal4UAS-binding Materials sites and 135 base pairs of the HSV TK gene upstream Phorbol ester (Sigma), EGF (B–D Biosciences), trichostatin A regulatory region linked to a firefly luciferase gene. To assay (TSA) (Biomol), H5106 (Bachem), rottlerin, bis-indolylmalei- ISRE-regulated luciferase reporter gene activity, a fragment mide I (BIM) and Go¨ 6976 (all from Alexis), and a rabbit encoding the ISG15 ISRE was transferred from a previously polyclonal antibody against the HA epitope (Santa Cruz) were described plasmid ISRE reporter (Improta and Pine, 1997)

Oncogene IFNaR2 receptor proteolysis AZM Saleh et al 7085 into pGL3 promoter (Promega) to create pGL3-ISRE-luc. (0.5 mg) plasmid DNA, using 2 ml of Lipofectamine and 3 mlof PGL3 promoter was used as a control in these experiments. PLUS reagent (Invitrogen), following the manufacturer’s Full-length IFNaR2 containing an HA epitope tag at the directions. After 1 day, cells were re-seeded on glass coverslips extreme carboxyl terminus was described previously (Nguyen in 3.5 cm dishes. The next day, cells adherent to the coverslips et al., 2002). This construct was transferred into pcDNA Zeo were fixed with 4% (w/v) paraformaldehyde in phosphate- 3.1 (À) for use in establishing the U5A stable transfectants buffered saline, counterstained with 2 mg Hoechst 33258/ml analysed in Figure 2b and c. A construct spanning the putative and visualized under a Nikon Eclipse E600 fluorescent ICD (residues 261–515), preceded by an initiator methionine, microscope equipped with a Spot RT digital camera and was prepared by PCR and cloned into pMT2T. Derivatives of accompanying software. IFNaR2 with deletions within the extracellular domain were prepared by PCR. For E1, PCR was used to join amino acid 220 to a FLAG-MluI epitope (DYKDDDDKTR). A second Reporter gene assays PCR amplification joined the FLAG tag to residue 45 at an CHO or HEK293T cells, plated in six-well cluster dishes EcoRV site. Thus, in E1, residues 33–219 are deleted and (2.3 Â 105 cells per well) were transfected via calcium phos- replaced by the indicated FLAG epitope. For E2, a similar set phate precipitates with pMT-I2-ECD-GV (5 or 1 mg, respec- of PCR fragments deleted residues 33–232 and inserted tively), pG4-TKluc (5 or 100 ng, respectively) and pRL-TK residues 43–44 (EcoRV site). (15 ng or 100 pg, respectively). In some cases, HEK293T cells were also transfected with PKCd-expressing plasmids (500 ng/ Protein extracts and immunoblotting well). U5A cells, plated in 24-well cluster dishes (1.2 Â 105 cells Cells expressing IFNaR2 derivatives were either previously per well), were transfected using Lipofectamine PLUS described (Nguyen et al., 2002), or generated by transiently (Invitrogen) with the 250 ng pGL3-ISRE-luc (or vector), transfecting HEK293T cells or stably transfecting U5A cells, 300 ng pMT2T-ICD (or vector) and 50 ng pRL-TK. At 2 days followed by selection in 75 mg/ml zeocin (Invitrogen). Com- after transfection, cell lysates were coordinately assayed for bined cytoplasmic and plasma membrane protein extracts were firefly luciferase and Renilla luciferase activities using a Dual prepared from NP-40 lysed cells as described previously Luciferase Reagent kit (Promega), according to the manufac- (Nguyen et al., 2002). Membrane preparations used in the in turer’s instructions. Activity, in relative luciferase units (RLU), vitro cleavage assay were prepared essentially as described by was calculated by dividing each determination of firefly others (Lee et al., 2002). In brief, 8 Â 107 cells were lysed by luciferase activity by the appropriate Renilla luciferase activity repeatedly passing the appropriate cell suspension through a value. Error bars correspond to the standard error of the mean. For experiments employing U2OS cells, 10 cm dishes syringe fitted with a 23-gauge needle (25 strokes). The lysate 5 was centrifuged at 1.5 Â 103 g to pellet unbroken cells and containing 4–6 Â 10 cells were transfected via calcium organelles. The supernatant was further centrifuged at phosphate precipitates, with either pSG424 (expressing 1.4 Â 104 g to pellet plasma membranes. This pellet was Gal4DBD), or pSG–Gal4–I2–ICD (expressing Gal4DBD resuspended in a small volume, incubated at either 4 or 371 fused to the IFNaR2 ICD) (5–10 mg), pG4-TKluc (5–10 mg) and then centrifuged at 2 Â 105 g. The final supernatant was and a constitutively expressed b-galactosidase gene (10 mg). used for immunoblotting. Cell fractions for immunoblotting Activity in RLU was calculated as above, using firefly were prepared similarly. In some cases, protein concentrations luciferase and b-galactosidase activities. were measured, using the BCA kit (Pierce), prior to electrophoresis. Protein electrophoresis and immunoblotting were performed as described previously (Nguyen et al., 2002). Acknowledgements We thank H Young and D Selkoe for cell lines; B Weinstein, J Chan and G Carpenter for plasmids; K Nastiuk for antibody Fluorescence microscopy and M Brunda and JRosa for IFN a. This work was supported U2OS cells (104 cells in individual wells of a 12-well cluster by a grant from the National Institutes of Health (CA56862) dish) were transfected with GFP–H2–ICD or GFP–I2–ICD to JJK.

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