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Vol. 16, Issue 11, 5061-5069, November 2005

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CD74 Is a Member of the Regulated Intramembrane Proteolysis-processed Protein Family

Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel

Submitted April 20, 2005; Revised August 9, 2005; Accepted August 10, 2005
Monitoring Editor: Peter Walter

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Quite a few regulatory proteins, including transcription factors, are normally maintained in a dormant state to be activated after internal or environmental cues. Recently, a novel strategy, requiring proteolytic cleavage, was described for the mobilization of dormant transcription factors. These transcription factors are initially synthesized in an inactive form, whereas "nesting" in integral membrane precursor proteins. After a cleavage event, these new active factors are released from the membrane and can migrate into the nucleus to drive regulated gene transcription. This mechanism, regulated intramembrane proteolysis (RIP), controls diverse biological processes in prokaryotes and eukaryotes in response to a variety of signals. The MHC class II chaperone, CD74 (invariant chain, Ii), was previously shown to function as a signaling molecule in several pathways. Recently, we demonstrated that after intramembranal cleavage, the CD74 cytosolic fragment (CD74-ICD) is released and induces activation of transcription mediated by the NF-B p65/RelA homodimer and the B-cell-enriched coactivator, TAF_II105. Here, we add CD74 to the growing family of RIP-processed proteins. Our studies show that CD74 ectodomain must be processed in the endocytic compartments to allow its intramembrane cleavage that liberates CD74 intracellular domain (CD74-ICD). We demonstrate that CD74-ICD translocates to the nucleus and induces the activation of the p65 member of NF-B in this compartment.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Quite a few regulatory proteins, including transcription factors, are normally maintained in a dormant state and are activated by internal or environmental cues. Recently, a novel strategy (regulated intramembrane proteolysis [RIP]), based on proteolytic cleavage, was discovered for the mobilization of dormant transcription factors. These transcription factors are synthesized initially as inactive, membrane-bound precursors. Once triggered, RIP proteins are cleaved within the plane of the membrane, and their cytosolic fragment migrates into the nucleus to drive transcription.

In general, in most RIP cases reported, cleavage proceeds through a two-step sequential proteolytic process. The first step involves the cleavage of the extracytoplasmic segment to shorten the ectodomain to <30 aa. This seems to be a requirement for the second proteolytic event, which occurs at the transmembrane domain. The initial shedding prepares the substrate for the intramembrane proteolysis such that the transmembrane region becomes accessible to the second protease, which then releases the product from the lipid bilayer. The released cytosolic fragment then migrates into the nucleus. Intramembrane cleaving proteases (I-CLiPs) catalyze peptide bond hydrolysis in the plane of cellular membranes. These proteases are thought to mediate RIP (Brown et al., 2000), and the reactions they catalyze are, in most cases, part of highly controlled processes. The family of I-CLiPs is growing, and at present, three distinct protease families have been shown to catalyze intramembrane proteolysis (Urban and Freeman, 2002; Weihofen and Martoglio, 2003; Lemberg and Martoglio, 2004). This tightly regulated mechanism guarantees controlled proteolysis in the plane of the membrane and prevents random degradation of membrane proteins (Brown et al., 2000; Hoppe et al., 2001; Urban and Freeman, 2002).

CD74 is a nonpolymorphic type II integral membrane protein; it has a short N-terminal cytoplasmic tail of 28 amino acids, followed by a single 24-aa transmembrane region. Alternative initiation of translation and differential splicing of the transcription products generate two different isoforms in mice (p31 and p41; Stumptner-Cuvelette and Benaroch, 2002). The CD74 chain was thought to function mainly as an MHC class II chaperone, which promotes ER exit of MHC class II molecules, directs them to endocytic compartments, prevents peptide binding in the ER, and contributes to peptide editing in the MHC class II compartment (Stumptner-Cuvelette and Benaroch, 2002). However, in addition to its function as a chaperone molecule, CD74 was recently shown to have a role as an accessory signaling molecule. An accessory role for CD74 was identified during T-cell responses through interactions with CD44 (Naujokas et al., 1993). Recently, CD74 was reported to be a high-affinity binding protein for the proinflammatory cytokine, macrophage migration-inhibitory factor (MIF), providing further evidence for a role in signal transduction pathways. MIF binds to the extracellular domain of CD74, and CD74 is required for MIF-mediated phosphorylation of the extracellular signal-regulated kinase-1/2 (ERK-1/2), cell proliferation, and prostaglandin E2 (PGE2) production (Leng et al., 2003). Besides its roles in inflammation and immunity, MIF is suggested to be involved in tumor cell growth and angiogenesis (Nishihira et al., 2003). Moreover, CD74 was shown to be directly involved in the maturation of follicular B-cells (Shachar and Flavell, 1996) and accumulation of marginal zone B-cells (Benlagha et al., 2004). The role of CD74 in follicular B-cell differentiation involves induction of a pathway leading to the activation of transcription mediated by the NF-B p65/RelA homodimer and its coactivator, TAFII105 (Matza et al., 2001). NF-B is activated by the intracellular domain of CD74 (CD74-ICD) that is liberated from the membrane. Amino acids 42–44 were found to be essential for this cleavage event (Matza et al., 2002).

The process of intramembrane cleavage followed by nuclear translocation and transcriptional activation is reminiscent of regulated intramembrane cleavage (RIP). Intramembrane cleavage of RIP-processed proteins is signal dependent; however, the natural ligand for CD74 in B-cells is still unknown.

To determine whether CD74 is processed by RIP, we followed the processing and cleavage of the CD74 extracellular domain, I-CLiP, the release of CD74-ICD, and its translocation to the nucleus. The results presented here show that arrival of CD74 to the endocytic compartments and the sequential proteolytic cleavages in these compartments are necessary for the intramembrane cleavage and for the release of CD74-ICD. CD74-ICD translocates to the nucleus to activate of NF-B, allowing B-cell maturation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cells
Spleen cells were obtained from various strains of mice at 6–8 wk of age as previously described (Shachar et al., 1995). All animal procedures were approved by the Animal Research Committee at the Weizmann Institute. B-cells were enriched as previously described (Flaishon et al., 2000; Becker-Herman et al., 2002). Cells were treated with either brefeldin A (BFA; 2.5 µg/ml), nocodazole (NOC; 2.5 µg/ml), monensin (Mon; 10 µM), leupeptin (LEU; 1 mM), and DAPT (WO 9822494; 50 µM) purchased from Sigma-Aldrich (St. Louis, MO) for 3 h, or (ZLL)2-ketone (10 µM), a kind gift from Dr. B. Martoglio.

Constructs and Molecular Cloning
CD74 LI7–8AA-GFP was constructed in pEGFP-C1 (Clontech, Palo Alto, CA), CD74 LI7-8AA myc and 1-82 LI7–8AA myc were constructed in pEF4/Myc-HisA (Invitrogen, Carlsbad, CA) using QuikChange Site-Directed Mutagenesis Kit (catalogue no. 200518, Stratagene, La Jolla, CA), with the following primers: 5' GGATGACCAACGCGACGCCGCCTCTAACCATGAACAGTTGCCC 3'; 5' GGGCAACTGTTCATGGTTAGAGGCGGCGTCGCGTTGGTCATCC 3'; GFP 1-197 in pEGFP-C1 (Clontech) was constructed using QuikChange Site-Directed Mutagenesis Kit, with the following primers: 5' GCCACTGGATATGTAAGACGAAGCTTCTGGCCTGG 3'; 5' CCAGGCCAGAAGCTTCGTCTTACATATCCAGTGGC 3'.

CD74 1–197 myc, CD74 1–160 myc, CD74 1–120 myc, and CD74 1–100 myc were constructed in pEF4/Myc-HisA (Invitrogen) as described (Matza et al., 2002) with the following primer sequences for cloning: 5' primer as described in Matza et al. (2002); 3' primers: 5' TCTGCAGAATTCCATGTCCAGTGGCTCTTTAGGTGG 3' for CD74 1–197 Myc; 5' TCTGCAGAATTCGTTCACGCCATCCATGGAGTTCTTAAG 3' for CD74 1–160 Myc; 5'TCTGCAGAATTCCATGTTGCCGTACTTGGTAACGTT 3' for CD74 1–120 Myc; 5' TCTGCAGAATTCTGGACGCATCAGCAAGGGAGTAGC 3' for CD74 1–100 Myc.

GFP CD74 1–100 were constructed in pEGFP-C1 (Clontech) plasmid as described in Matza et al. (2002). The 5' primer was described in Matza et al. (2002); 3' primer: 5' TCTGGTGGATCCTATGGACGCATCAGCAAGGGAGTAGC 3' for GFP CD74 1–100.

The Xpress-CD74 construct was prepared by cloning to pEF4/Xpress-HisA (Invitrogen), using cloning primer sequences as follows: 5' primer at position 1 of CD74: 5'-CCTAGGATCCATGATGACCAACGCGACCTCATCTCT-3'; 3' primer at the last amino acid of CD74 (FL): 5'-TGCAGAATTCCTCACAGGGTGACTTGACCCAGTTC-3'.

CD74 1–42 nuc and CD74 1–42 mito constructs were prepared by cloning to pEF/myc/nuc and pRE/myc/mito plasmids (Invitrogen), using cloning primer sequences as follows: 5' primer at position 1 of CD74: 5'-CGCTGCAGGATGACCAACGCGACCTCATCTCT-3'; 3' primer at position 42 of CD74: 5'-CTGCGGCCGCCACCAGGACAGAGACACCGGTGT-3'.

For all the constructs, the PCR products were separated by agarose electrophoresis to ensure the correct size and purified from the gel using Wizard PCR Preps (Promega, Madison, WI). The products and the cloning plasmids were digested by PstI and NotI (NEB) for pEF/myc/nuc and pRE/myc/mito plasmids or by BamHI (New England Biolabs, Beverly, MA) followed by EcoRI (Amersham Pharmacia Biotech, Piscataway, NJ) for pEF4/Xpress-HisA, and the products were ligated into the appropriate plasmid vector. Clones were subjected to automated DNA sequencing by standard protocols using an ABI377 machine (PE Biosystems, Foster City, CA). The full sequence of all CD74 inserts was verified with no errors in the sequence.

Rous sarcoma virus (RSV)-Renilla luciferase plasmid was a generous gift from M. Walker (Weizmann Institute of Science, Rehovot, Israel).

Cell Transfection
HEK 293 cells were seeded in a 10-cm² dish. Transfections were performed using the standard CaPO₄ method as described previously (Matza et al., 2002). A total of 5 µg DNA (FL CD74) or 1 µg (1–82 Myc) were used per 10-cm² dish. At 24-h posttransfection, cells were treated with either BFA (2.5 µg/ml), NOC (2.5 µg/ml), Mon (10 µM), chloroquine (25 µM/100 µM), or LEU (1 mM) for 3 or 5 h.

Purified CD74–/– B-cells were incubated with 50 µg/ml LPS from Salmonella typhosa (Sigma). After 48 h, the cells were washed with RPMI media and transfected with TransFast transfection reagent (Promega) using 12 µl/4 µg DNA (2 µg CD74 construct + 2 µg empty vector or 4 µg empty vector) according to the manufacturer's directions. The cells were collected after 48 h and analyzed for their cell surface marker expression by FACS analysis. The percentage increase of IgD+ cells was calculated by subtracting the X mean fluorescence value of IgD+ staining of empty expression plasmid and dividing it to the same X mean value multiplied by 100%.

Cell Fractionation
Full fractionation: The fractionation method was adopted from Wang et al. (1994). Transfected 293 cells were disrupted by incubation in 5 vol of low-salt buffer supplemented with protease inhibitors (buffer C) for 30 min, followed by passage 10 times through a 25-gauge needle. The lysate was centrifuged at 1000 x g for 10 min to obtain the crude nuclei. The supernatant was further centrifuged at 100,000 x g for 1 h to obtain a cytosolic fraction and a crude membrane pellet. This pellet was washed in a 500 mM NaCl buffer with protease inhibitors (buffer D) to obtain a membrane fraction.

The low-centrifugation nuclear pellet was washed three times with buffer C and then incubated with buffer D for 30 min followed by 20,000 x g centrifugation for 20 min to obtain nuclear extract and nuclear debris fractions. Both pellet fractions were solubilized in 10 mM Tris-HCl (pH 6.8), 0.1 M NaCl, and 1% (vol/vol) SDS. All the fractions were supplemented with SDS loading buffer and boiled for 10 min.

Separation between membrane and supernatant fractions: HEK 293 cells were grown to confluency on 10-cm tissue culture dishes, rinsed once with cold phosphate-buffered saline (PBS) and once with cold 20 mM Tris-Cl (pH 7.4), 1 mM MgCl₂, 2 mM EGTA, 0.1 mM sodium orthovanadate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (hypotonic lysis buffer; HLB), and lysed by Dounce homogenization after swelling in 700 µl of HLB per 10-cm dish. Lysates were centrifuged for 45 min at 10,000 rpm in an Eppendorf microcentrifuge at 4°C to separate the particulate and soluble fractions. Soluble fractions were lyophilized and resuspended in a small volume.

Luciferase Assay for Monitoring NF-B Activation
Subconfluent 293 cells were transfected in a 24-well plate using a total of 1 µg plasmid; empty or CD74 expression vectors (25 ng) were added together with 20 ng of the Gal4 luciferase reporter, 0.5 ng of DBD fusion plasmids, and 1 ng of RSV-Renilla luciferase. The total amount of DNA was kept constant by adding pBabe vector. After transfection, cells were incubated 24 h and then harvested, and their luciferase and Renilla luciferase activities were measured.

Preparation of Cell Extracts and Western Blot Analysis
Cells were pelleted and incubated with 50 µg/ml digitonin (Sigma) and the pellet was then lysed as previously described (Shachar et al., 1995). For hot SDS-cells were collected 48 h posttransfection and cell lysates were prepared as described previously (Erickson and Blobel, 1979). Lysates were resolved by SDS-PAGE or Tricine gels and electroblotted onto nitrocellulose. Tricine-SDS-PAGE (16%, wt/vol) and transfer to membranes was performed as previously described (Schagger and von Jagow, 1987). The blots were blocked with 10% (vol/vol) skim milk for 1 h and then probed for 1 h with IN1 (anti-CD74 cytoplasmic tail mAb) antibodies followed by washing and 1-h incubation with horseradish peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and peroxidase visualization by enhanced chemiluminescence (Amersham).

Fluorescence Microscopy
HEK 293 cells were plated on a coverslip. After 24 h, the cells were transfected as described above. After an additional 48 h, the cells were fixed with 3% PFA and were mounted on glass coverslip with Mowiol 4–88 (Calbiochem, La Jolla, CA).

Staining of the endosomes was performed with LysoTracker Red (Molecular Probes, Leiden, The Netherlands) according to the manufacturer's directions.

View larger version (38K): [in this window] [in a new window]   Figure 1. Diagram of tagged CD74 constructs used in this study.

Immunofluorescence Microscopy
Transfected cells seeded on coverslips were fixed with 3% PFA and permeabilized with 0.4% Triton in 3% PFA. The cells were blocked with 10% fetal calf serum and incubated 1 h with IN1 antibody, followed by washing and incubation with CY-conjugated anti-rat antibody and DAPI. Then the cells were mounted on slides and analyzed as above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Processing of CD74 in the Endocytic Compartments Is Required for Its Intramembrane Cleavage
CD74 undergoes several proteolytic events in the endocytic compartments, resulting in the removal of the bulk of the lumenal domain and formation of a membrane-bound truncated protein (Stumptner-Cuvelette and Benaroch, 2002). To determine whether processing of the CD74 extracytoplasmic domain in the endocytic compartments is required for its intramembrane proteolytic cleavage, we blocked the export of CD74 to these endocytic compartments and analyzed the release of CD74-ICD. BFA inhibits protein transport resulting in their accumulation in the ER (Klausner et al., 1992; see Figure 2A). HEK 293 cells, which do not express endogenous CD74 or MHC class II molecules were transfected with the full length p31 (FL) CD74-myc construct (Figure 1). The CD74-transfected cells were incubated in the presence or absence of BFA for 3 h before their harvest; cell lysates were separated on Tricine gels, and the levels of CD74-ICD were analyzed by Western blot analysis probing with the IN1 antibody, which recognizes the CD74 cytosolic domain. As can be seen in Figure 2B, treatment with BFA resulted in a reduction of CD74-ICD release, suggesting that export from the ER is required for CD74 cleavage. However, as reported previously, BFA induces retrograde transport of proteins from the Golgi to the ER (Klausner et al., 1992). To determine whether this influx of Golgi proteins to the ER might influence CD74-ICD cleavage and whether other Golgi proteins might be responsible for the reduced liberation of CD74-ICD, the retrograde transport was blocked by NOC, an inhibitor of microtubule polymerization. As can be seen in Figure 2B, although NOC itself did not influence CD74-ICD release, CD74-ICD cleavage was largely inhibited in cells treated with both BFA and NOC. To determine whether these inhibitors similarly influence CD74-ICD release in primary B-cells, splenocytes were incubated in the presence or absence of BFA and NOC for 3 h. As can be seen in Figure 2C, although NOC treatment did not affect CD74-ICD release, the liberation of CD74-ICD was dramatically inhibited in the BFA-treated cells (in the presence or absence of NOC), showing that CD74 intramembrane cleavage is not catalyzed by ER and Golgi enzymes.

View larger version (51K): [in this window] [in a new window]   Figure 2. Arrival to the endocytic compartments is essential for CD74 intramembrane cleavage. (A) Fluorescence microscopy of 293 cells transfected with the GFP FL p31 CD74 chimera. At 24 h posttransfection, cells were incubated in the presence or absence of brefeldin A (BFA; 2.5 µg/ml) and in the presence or absence of NOC (2.5 µg/ml). (B and C) Western blots (B) FL-CD74 was transfected into 293 cells, which were then treated with BFA (2.5 µg/ml) in the presence or absence of NOC (2.5 µg/ml). Total lysates were separated on Tricine gel and analyzed with IN1 rat monoclonal antibodies that recognize the CD74 cytosolic domain. Ratio calculation: The intensity of CD74-ICD band in each treatment was divided by the intensity of a nonrelevant band in each lane. The CD74-ICD ratio in the absence of any treatment was normalized to 1 and the ratio for each treatment was calculated as the intensity of the treatment sample relative to 1. The results presented are representative of three different experiments. (C) Total splenocytes were treated with BFA (2.5 µg/ml), NOC (2.5 µg/ml), LEU (1 mM), and with MON (10 µM) for 3 h; lysates were separated on Tricine gel and analyzed with IN1. The results presented are representative of five different experiments.

View larger version (39K): [in this window] [in a new window]   Figure 3. Processing in the endocytic compartments is essential for CD74 intramembrane cleavage. (A) Fluorescence microscopy of 293 cells transfected with GFP FL and GFP FL LI 7,8 AA (green) shown together with the endocytic compartment staining (red). (B) Western blot analysis of 293 cell lysates transfected with FL myc and FL LI7,8AA Myc; at 24 h posttransfection, lysates were separated on Tricine gel and analyzed with IN1. (C and D) FL-myc was transfected into 293 cells, which were treated with MON (10 mM) (C) and with chloroquine (25 and 100 µM; D) for 3 h. Total lysates were separated on Tricine gel and analyzed with IN1. The intensity of the CD74-ICD band after each treatment was divided by the intensity of a nonrelevant band in each lane. The CD74-ICD ratio in the absence of any treatment was normalized to 1 and the ratio for each treatment was calculated as the intensity of the treatment sample relative to 1. The results presented are representative of three different experiments.

In the endocytic compartments, CD74 is processed by a series of enzymes to generate a fragment of 82 aa, lacking most of its lumenal sequence. To determine whether this processing is essential for the release of CD74-ICD, the proteolytic activity in these compartments was inhibited using Mon, which inhibits endosomal activity by blocking acidification of the lumenal compartments (Dinter and Berger, 1998). HEK 293 cells transfected with the FL Myc construct (Figure 1) and primary B splenocytes were incubated in the presence or absence of Mon for 3 h before their harvest.

Lysates were separated on Tricine gel and the liberation of CD74-ICD was monitored by Western blotting. Treatment with Mon largely inhibited CD74-ICD cleavage in both 293 cells (Figure 3C) and in primary B-cells (Figure 2C), suggesting that exit from the TGN and the enzymatic activity in the endocytic compartments are essential for CD74 intramembrane cleavage. To specifically inhibit the activities of the endocytic compartments and the processing of CD74 in these compartments, we used an acidotropic agent, chloroquine, which blocks endosomal acidification (de Duve et al., 1974). Primary B splenocytes were incubated in the presence or absence of chloroquine for 3 h before their harvest. Lysates were separated on Tricine gel and the formation of CD74-ICD was monitored. As shown in Figure 3D, treatment with chloroquine resulted in a dramatic inhibition of CD74 processing and the formation of CD74-ICD was largely blocked. Thus, processing of CD74 in the endocytic compartments is essential for its transmembrane cleavage and CD74-ICD release.

To further demonstrate the importance of CD74 lumenal domain processing in CD74-ICD release, we directly inactivated proteases involved in this process using LEU. LEU is an inhibitor with broad specificity for cysteine proteases and was previously shown to interfere with the processing of CD74, resulting in accumulation of the CD74 intermediate fragments (Villadangos et al., 1997). Splenocytes were treated for 3 h in the presence or absence of LEU and the formation of CD74-ICD was followed by Western blot analysis. As seen in Figure 2C, treatment with LEU resulted in accumulation of the processing products, and inhibition of CD74-ICD release. Hence, processing of the CD74 lumenal domain is essential for the release of its cytosolic fragment to the cytoplasm.

CD74 Lumenal Domain Regulates Its Intramembrane Cleavage and NF-B Activation
To further demonstrate that the processing of CD74 lumenal domain in the endocytic compartment is essential for its intramembrane cleavage, we followed the release of CD74-ICD in various constructs containing sequential deletions of CD74 sequences and analyzed the release of CD74 cytosolic domain in each truncated construct. Within the endosomes, CD74 lumenal domain undergoes a stepwise proteolytic cleavage that yields progressively smaller fragments. An initial cleavage event generates the LIP22 fragment (160 aa), which is then cleaved to generate the LIP10 fragment (100 aa) and to enable the liberation of CLIP. We generated CD74 constructs of different sizes, including several that mimic the natural cleavage products of the lumenal domain. CD74 FL, 1–197, 1–160, 1–120, 1–100, and 1–82, C-terminally fused to GFP or Myc epitopes (Figure 1) were transfected into HEK 293 cells. To determine the role of CD74 lumenal domain on its intramembrane cleavage, we followed the localization of CD74-ICD -GFP in cells transfected with CD74 truncated mutants fused to GFP (Figure 1). As can be seen in Figure 4 GFP-FL and GFP-1–197 (Figure 4A) and FL-Myc and 1–160-Myc (Figure 4B) were mostly localized in the endocytic compartments, as previously described (Pieters et al., 1993). However, removal of the bulk of the lumenal domain, resulted in cytoplasmic localization of CD74 cytosolic domain (Figure 4, A and B) as was previously shown for the GFP 1–82 construct (Matza et al., 2002). Thus, deletion of the lumenal domain allows a more efficient release of its cytosolic fragment to the cytoplasm.

View larger version (55K): [in this window] [in a new window]   Figure 4. Localization of CD74 cytosolic domain. (A) Fluorescence microscopy of 293 cells transfected with the GFP FL, GFP 1–197, GFP 1–100, and GFP 1–82 chimera (green) together with the endocytic compartment staining (red). The results presented are representative of three different experiments. (B) 293 cells expressing the indicated forms of CD74, were preincubated for 40 min at 4°C with an EGF conjugated to Alexa Fluor 488. Thereafter, cells were incubated for 20 min at 37°C, permeabilized, and stained with an anti-CD74 antibody and detection using a CY-conjugated secondary antibody. The distribution of fluorescent EGF and CD74 is shown, along with merge panels depicting both signals.

To further establish the inhibitory role for CD74 lumenal domain, Myc-transfected cells were collected, their lysates were separated on Tricine gels, and the levels of CD74-ICD in transfected cells were analyzed by Western blot analysis with the IN1 antibody. Although the release of CD74-ICD from FL, 1–197, 1–160 Myc (Figure 5A), and 1–120 Myc (Figure 5B) constructs was at barely detectable levels, a dramatic elevation in the liberation of this fragment was observed after transfection of the 1–100 and 1–82 constructs (Figure 5, A and B). Together, these results indicate that the processing of CD74 in the endocytic compartments enables its intramembrane cleavage and the liberation of CD74-ICD.

View larger version (40K): [in this window] [in a new window]   Figure 5. CD74 lumenal domain regulates its intramembrane cleavage in the endocytic compartments and NF-B activation. (A and B) 293 cells were transfected with CD74 full length, CD74 1–197, CD74 1–160, CD74 1–120, CD74 1–100, and CD74 1–82. Myc tagged constructs were separated on Tricine gels and analyzed with anti CD74. Ratio calculation: the intensity of the CD74-ICD band in each construct was divided by the intensity of a nonrelevant band in each lane. The CD74-ICD ratio in the full-length (A) or 1–120 (B) transfection was normalized to 1 and the ratio of CD74-ICD in each construct was calculated as the intensity relative to 1. (C–E) 1–82 and 1–60 myc constructs were transfected into 293 cells and treated with MON (10 µM; C). 293 cells were transfected with 1–82 myc construct and treated with LEU (1 mM) for 3 h (D). 293 cells were transfected with 1–82 and 1–82 LI 7,8 AA (E). Total lysates were separated on Tricine gel and analyzed with IN1 rat monoclonal antibodies, which recognize the CD74 cytosolic domain. Ratio calculation: the intensity of CD74-ICD band in each treatment was divided by the intensity of a nonrelevant band in each lane. CD74-ICD ratio in the absence of any treatment was normalized to 1, and the ratio for each treatment was calculated as the intensity for that treatment relative to 1. The results presented are representative of three different experiments. (F) Nonsaturating amounts of G4-p65 TA1 (the TA1 activation domain of p65/RelA fused to the GAL4 DBD) and luciferase reporter gene containing the Gal4 binding site were transfected into 293 cells in the presence of 25 ng of FL, 1–160, 1–100, and 1–82 Myc constructs. Luciferase activities were normalized to the activity of cotransfected RSV promoter-driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of each CD74 construct relative to the activity of the empty plasmid. The graph represents the average of five independent experiments.

Previously, we showed that CD74 induces B-cell maturation by activating the TAF_II105–NF-B-dependent transcription program (Matza et al., 2001). CD74 induces the activation domain of the p65/RelA protein, a process that is essential for maturation of B-cells. To determine whether processing of the CD74 lumenal domain, required for CD74-ICD release, is also essential for NF-B activation, a fusion of the C-terminal transactivation domain of p65/RelA and the DNA-binding domain of the yeast transcription factor, Gal4, was transfected into 293 cells along with a luciferase reporter containing the Gal4-binding sites, in the presence of various CD74 truncated plasmids (Matza et al., 2001) and the RSV promoter, which was used as a reference. As can be seen in Figure 5F, augmented NF-B activation was detected in constructs lacking the bulk of the lumenal domain (CD74 1–100myc 1–82myc). Thus, the natural processing of CD74 lumenal domain prepares the molecule for efficient intramembrane cleavage. The release of CD74-ICD induces NF-B-mediated transcription.

The I-CLiP Involved in CD74 Intramembrane Cleavage
Intramembrane proteolysis is a widely conserved mechanism for controlling diverse biological processes (Brown et al., 2000; Kopan and Goate, 2000). Several protease families are currently known to catalyze intramembrane proteolysis: S2P, Rhomboid and the presenilins (PS), and SPP-type aspartic proteases (Weihofen and Martoglio, 2003). Presenilins play a catalytic role in the -secretase complex required for regulated intramembrane proteolysis (Haass and Steiner, 2002). To determine which protease is involved in CD74 cleavage, we analyzed whether the intramembrane proteolytic processing of CD74 is dependent on -secretase activity. HEK 293 cells express endogenous PS-dependent -secretase activity and have been widely used to investigate PS-dependent processing of APP (De Strooper et al., 1998; Naruse et al., 1998), CD44 (Lammich et al., 2002; Murakami et al., 2003), ErbB-4 (Ni et al., 2001), E-cadherin (Marambaud et al., 2002), and Notch (Lammich et al., 2002). HEK 293 cells were cotransfected with a FL CD74 construct and a dominant negative construct of presenilin (DN-PS1). Cells were then separated into membrane and soluble fractions. The soluble fraction was concentrated and analyzed for the presence of the cleaved cytoplasmic domain. As can be seen in Figure 6A, in the presence of DN-PS1, although, in accord with previous studies (Wilhelmsen and van der Geer, 2004), we did not detect a massive accumulation of the uncleaved membranal protein, very little of the CD74-ICD was released into the cytosol. To further follow the involvement of presenilin in CD74 intramembrane cleavage, we used the -secretase/PS1 specific inhibitor, DAPT (WO 9822494). HEK 293 cells were transfected with a FL CD74 construct and incubated in the presence or absence of DAPT for 3 h. Cells were then separated into membrane and soluble fractions and analyzed for the presence of the cytoplasmic domain. As can be seen in Figure 6B, although we did not detect a massive accumulation of the uncleaved membranal protein, a dramatic inhibition of CD74-ICD release was detected in DAPT-treated cells.

View larger version (41K): [in this window] [in a new window]   Figure 6. -secretase cleaves CD74. (A) 293 cells were cotransfected with FL-CD74 Myc and a dominant negative construct of presenilin (PS1). Lysates were separated into a particulate and cytosolic fractions, separated on Tricine gel and analyzed with IN1 antibody. The results presented are representative of five different experiments. (B and C) 293 cells were transfected with FL-CD74 Myc (B) or 1–82 Myc (C) constructs and treated with or without DAPT (50 µM; B and C) or (ZLL)2-ketone (10 µM; C) for 5 h. Lysates were separated into a particulate and cytosolic fractions, separated on Tricine gel, and analyzed with IN1 antibody. The results presented are representatives of five different experiments. CD74-ICD ratio in the absence of any treatment was normalized to 1, and the ratio for each treatment was calculated as the intensity for that treatment relative to 1.

Recently, the presenilin-related aspartic protease, SPP, was described (Weihofen et al., 2002). Like presenilins, SPP contains the aspartic protease motifs, YD and LGLGD. The striking difference between SPP and presenilins is the opposite topology of the transmembrane regions that they cleave. Therefore it was suggested that each particular I-CLiP only attacks substrates of one particular orientation, e.g., either type I or type II, but not both (Weihofen and Martoglio, 2003; Nyborg et al., 2004). Because CD74 is a type II protein, the involvement of SPP and not presenilin in its intramembrane cleavage was expected. We therefore attempted to inhibit CD74 cleavage using the specific SPP inhibitor, (Z-LL)2 ketone (Weihofen et al., 2000; Weihofen and Martoglio, 2003). It was previously shown that the (Z-LL)2 ketone can also inhibit cathepsin S (Weihofen et al., 2000), an enzyme that is involved in CD74 processing in the endocytic compartment. Our studies show here that processing in the endocytic compartments regulates CD74-ICD release; therefore, the effect of (Z-LL)2 ketone was studied on the truncated CD74 amino acid 1–82 fragment, which is the final product of CD74 processing in the endocytic compartments and not a substrate for cathepsin S. HEK 293 cells were transfected with the 1–82 myc construct and incubated in the presence or absence of DAPT or (Z-LL)2 ketone. Cells were then separated into membrane and soluble fractions and analyzed for the presence of the cytoplasmic domain. As shown in Figure 6C, although DAPT dramatically downregulated CD74-ICD release, (Z-LL)2 ketone did not inhibit the release of this fragment. Thus, these data suggest that the cleavage reaction is apparently mediated by the -secretase/presenilin complex.

View larger version (28K): [in this window] [in a new window]   Figure 7. CD74-ICD translocates to the nucleus to activate NF-B. (A) Immunofluorescence microscopy of 293 cells transfected with 1–82 (1) myc or 1–42 myc (2 and 3). After transfection, cells were incubated 48 h, fixed, permeabilized, and stained with IN1 antibody followed by CY3-conjugated anti-rat antibody (1 and 2). The stained cells were analyzed by fluorescence microscopy. In (3), DNA was visualized using DAPI staining of the same cells (blue). (B) Cells were transfected with 1–42 myc and fractionated 48 h later. Equivalent cell numbers from each fraction were loaded on tricine gel. 1–42 was detected by Western blot analysis with IN1. The blot was then stripped and analyzed with anti--tubulin antibody as a marker for cytosolic fraction and with anti-SP1 for the nuclear fraction.

CD74-ICD Translocates to the Nucleus
CD74-ICD activates NF-B-mediated transcription via its effect on the transactivation domain of p65/RelA, while having no effect on the nuclear translocation of p65 (Matza et al., 2001). To determine whether CD74-ICD translocates to the nucleus, we searched for the presence of CD74-ICD in this compartment.

We have previously shown that CD74 cleavage occurs at amino acids 42–44. Moreover, the 1–42 truncated construct (tagged with Myc epitope, 1–42 myc, Figure 1) was shown to induce NF-B activation and B-cell maturation (Matza et al., 2002). Thus, to determine whether CD74-ICD translocates to the nucleus, we followed the subcellular localization of the 1–42 construct. To this end, we analyzed the localization of 1–42 myc construct (Figure 1) in HEK 293-transfected cells by indirect immunofluorescent staining with IN1 antibody (which recognizes the cytosolic domain of CD74). As can be seen in Figure 7A, although the 1–82 molecule is mostly detected in the cytosol, the 1–42 fragment, in contrast, exhibited apparent nuclear staining, along with cytosolic staining, suggesting that at least a portion of the CD74 ICD enters the nuclear compartment. To confirm this result, we performed a full biochemical fractionation of 293 cells, transfected with the 1–42 construct. In this procedure, the nuclei are precipitated from the disrupted cells by low-speed centrifugation and proteins are extracted from them by high-salt treatment, and the supernatant is separated into membrane and cytosol fractions by 100,000 x g centrifugation. As seen in Figure 7B, the 1–42 fragment was dispersed in the membrane, nuclear debris, cytosolic and nuclear fractions, indicating that CD74-ICD translocates to the nucleus.

To determine whether this nuclear import is essential for the functionality of CD74-ICD, a nuclear localization signal was conjugated to the 1–42 fragment, creating the 1–42 nuc construct (Figure 8A). As a control, an additional construct was generated, CD74 1–42 linked to a mitochondrial targeting signal (1–42 mito; Figure 8A). The predicted localization of each construct was verified by immunofluorescence of transfected cells using IN1 antibody (Figure 8B). Indeed, 1–42 nuc was found almost totally in the nucleus, whereas the staining of 1–42 mito-transfected cells showed mitochondrial-like appearance.

View larger version (22K): [in this window] [in a new window]   Figure 8. CD74-ICD activates NF-B in the nucleus. (A) Schematic illustration of the constructs of CD74 1–42, 1–42 nuc, and 1–42 mito. (B) HEK 293 cells were transfected with 1–42 Myc, 1–42 nuc, or 1–42 mito and the localization of CD74 was analyzed by immunofluorescence as described above. DNA was visualized using DAPI staining of the same cells (blue). (C) Subsaturating amounts of G4-p65 TA1 (the TA1 activation domain of p65/RelA fused to the Gal4 DNA-binding domain) and Gal4 reporter plasmid were transfected into HEK 293 cells along with increasing amounts of 1–42 myc, 1–42 nuc, or 1–42 mito plasmids. Luciferase activity was measured, and activation fold was calculated as described for Figure 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Quite a few regulatory proteins, including transcription factors, are normally kept in a dormant state to be activated in response to internal or environmental cues. Recently, a novel strategy, requiring proteolytic cleavage, was described for the mobilization of dormant transcription factors. These transcription factors are initially synthesized in an inactive form, while "nesting" in integral membrane precursor proteins. After a cleavage event, these new active factors are released from the membrane and can migrate into the nucleus to drive regulated gene transcription. This mechanism, RIP, is known for its conservation in several systems and its ability to control diverse biological processes in prokaryotes and eukaryotes in response to a variety of signals (Brown et al., 2000; Hoppe et al., 2001; Urban and Freeman, 2002).

In general, in most of RIP cases reported, cleavage proceeds in a two-step sequential proteolytic process. The first step involves the cleavage of the extracytoplasmic segment to shorten the ectodomain to <30 aa. This seems to be a requirement for the second proteolytic event, which occurs at the transmembrane domain. It appears that the initial shedding prepares the substrate for the intramembrane proteolysis such that the transmembrane region becomes accessible to the second protease, which releases the product from the lipid bilayer toward the cytosol. This mechanism, when regulated, guarantees controlled proteolysis in the plane of the membrane and prevents random degradation of membrane proteins (Brown et al., 2000; Hoppe et al., 2001; Urban and Freeman, 2002).

Some common features of RIP have shown remarkable similarities to the behavior of CD74. CD74 undergoes several proteolytic events in the endocytic compartments, resulting in the removal of the bulk of the lumenal domain and formation of a membrane-bound 1–82 truncated protein (Stumptner-Cuvelette and Benaroch, 2002). Here, we show that arrival to the endocytic compartments and a series of proteolytic cleavages in these compartments are necessary for the release of CD74-ICD from the membrane. Thus, blocking the arrival to the endocytic compartments and inhibitors that block the proteolytic events there arrest the cleavage event. Moreover, analysis of CD74 truncated constructs that represent various proteolytic products of the CD74 lumenal domain, including naturally processed CD74 fragments, indicate that the shedding of the lumenal domain in the endocytic compartments prepares the molecule for its transmembrane cleavage, which is essential for the activation of NF-B. Once the CD74 lumenal domain has been removed, CD74 can serve as a substrate for I-CLiP cleavage, a process that occurs exclusively in the endocytic compartments. Thus, transport to the endocytic compartment is essential for both CD74 processing and for its intramembrane cleavage. The removal of the CD74 lumenal domain therefore appears to be the first step required for the release of the N-terminal cytosolic fragment, leading to NF-B activation. To further understand the transcriptional function of CD74, it will be essential to determine whether the ectodomain shedding of CD74, like other RIP proteins, is signal dependent.

In most RIP proteins, the released cytosolic fragments translocate to the nucleus to elicit their biological responses. We therefore searched for the presence of CD74-ICD in the nucleus, by following the cellular localization of the aa 1–42 fragment. The immunostaining and biochemical fractionation of cells transfected with the 1–42 construct have revealed that this fragment can be detected in the various fractions, membrane, cytosolic and nuclear, confirming that CD74-ICD is released from the membrane to the cytosol and that a portion of this proteolytic product translocates to the nucleus.

Finally, to determine whether nuclear translocation is essential for the functionality of CD74 in NF-B activation, we followed the activity of NLS-tagged CD74-ICD. Although amino acids 1–42 were distributed in the cytosol and the nucleus, NLS-tagged CD74-ICD was found mostly in the nucleus. This peptide was found to be far more active in elevation of NF-B-mediated transcription than the naturally distributed 1–42. This effect on NF-B activity was directed to the transactivation domain of p65, as was previously shown for CD74 and CD74-ICD (Matza et al., 2001, 2002). Taken together, these results show that translocation of CD74-ICD to the nucleus is essential for its modulation of p65 transactivation function, suggesting that CD74-ICD indeed acts in the nucleus.

To conclude, our studies add CD74 to the growing family of RIP proteins and show that the roles of CD74 as a chaperone and as a signaling molecule are intertwined in a tightly regulated chain of command.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We gratefully acknowledge members of the Shachar lab for discussions and comments on this manuscript. This research was supported by The Israel Science Foundation founded by the Academy of Sciences and Humanities and the Minerva Foundation. I.S. is the incumbent of the Alvin and Gertrude Levine Career Development Chair of Cancer Research.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–04–0327) on August 17, 2005.

^* These authors contributed equally to this work.

Address correspondence to: Idit Shachar (idit.shachar{at}weizmann.ac.il).

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