CIITA Activity Through Phosphorylation CIITA

Serine Residues 286, 288, and 293 within the CIITA: A Mechanism for Down-Regulating CIITA Activity through Phosphorylation

This information is current as Susanna F. Greer, Jonathan A. Harton, Michael W. Linhoff, of October 2, 2021. Christin A. Janczak, Jenny P.-Y. Ting and Drew E. Cressman J Immunol 2004; 173:376-383; ; doi: 10.4049/jimmunol.173.1.376 http://www.jimmunol.org/content/173/1/376 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Serine Residues 286, 288, and 293 within the CIITA: A Mechanism for Down-Regulating CIITA Activity through Phosphorylation1

Susanna F. Greer,* Jonathan A. Harton,* Michael W. Linhoff,* Christin A. Janczak,† Jenny P.-Y. Ting,2* and Drew E. Cressman2,3†

CIITA is the primary factor activating the expression of the class II MHC genes necessary for the exogenous pathway of Ag processing and presentation. Strict control of CIITA is necessary to regulate MHC class II gene expression and induction of an immune response. We show in this study that the nuclear localized form of CIITA is a predominantly phosphorylated form of the protein, whereas cytoplasmic CIITA is predominantly unphosphorylated. Novel phosphorylation sites were determined to be located within a region that contains serine residues 286, 288, and 293. Double mutations of these residues increased nuclear CIITA, indicating that these sites are not required for nuclear import. CIITA-bearing mutations of these serine residues significantly increased endogenous Downloaded from MHC class II expression, but did not significantly enhance trans-activation from a MHC class II promoter, indicating that these phosphorylation sites may be important for gene activation from intact chromatin rather than artificial plasmid-based promoters. These data suggest a model for CIITA function in which phosphorylation of these specific sites in CIITA in the nucleus serves to down-regulate CIITA activity. The Journal of Immunology, 2004, 173: 376–383.

he major histocompatibility CIITA plays a pivotal role in onstrated that although CIITA itself does not bind DNA, it con- http://www.jimmunol.org/ initiating immune responses by activating the expression tains a potent trans-activation domain in the N terminus (15, 16). T of the MHC class II genes and associated molecules (1– CIITA also contains a variety of other recognizable motifs, includ- 6). MHC class II proteins facilitate the presentation of exogenously ing proline-serine-threonine-rich domains (17), a GTP-binding do- derived antigenic peptides on the surface of APCs, leading to rec- main (GBD)4 (18), and three nuclear localization signals (NLSs) ognition by CD4ϩ T cells and subsequent activation of the cellular (19–22). Loss of GTP binding prevents translocation of CIITA components of the immune system. Expression of MHC class II into the nucleus. Although the GBD is not an NLS, binding of GTP genes is constitutive on B cells, monocytes, macrophages, and den- to this region probably causes a conformational change, permitting dritic cells and is inducible on most other cell types in response to nuclear translocation (18). by guest on October 2, 2021 cytokines such as IFN-␥ and TNF-␣. Both constitutive and induc- Once in the nucleus, CIITA activates the expression of a variety ible expressions of these genes require CIITA, whereas loss of of genes, leading to MHC class II Ag surface presentation (5, 6). CIITA results in a severe immunodeﬁciency characterized by com- The precise mechanisms regulating CIITA activity and MHC class plete loss of MHC class II-mediated Ag presentation (7, 8). II expression have been areas of recent intense interest. It has Within the nucleus, CIITA interacts with a variety of transcrip- previously been shown that CIITA activity is inducible by IFN-␥, tion factors and coactivators such as regulatory factor X (RFX)5, enhanced through association with mono-ubiquitin, and down-reg- NF-Y, CREB, and CREB binding protein at the promoter regions ulated by the actions of histone deacetylases 1 and 2 (23–25). of target genes (9–14). CIITA acts as a molecular scaffold to sta- Although CIITA acts primarily in the nucleus, the CIITA protein bilize the interactions of these requisite MHC class II transcription has been found to localize to both the cytoplasm and nucleus and factors with the conserved MHC class II promoter motifs X1, X2, to undergo nuclear import (19–21, 26). The purpose of this dual and Y, and is therefore critical for the formation of the MHC class subcellular localization remains to be determined. Treatment of II enhanceosome complex. Structure-function studies have dem- cells with leptomycin B, an inhibitor of nuclear export, causes accumulation of CIITA in the nucleus. Export of CIITA is probably mediated by one or more nuclear export signals and has re- *Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599; and †Department cently been shown to be regulated in part by the GTP-binding of Biology, Sarah Lawrence College, Bronxville, NY 10708 domain (27). The possibility therefore remains that nuclear export Received for publication March 4, 2004. Accepted for publication April 19, 2004. of CIITA plays a role in down-regulating its activity and MHC The costs of publication of this article were defrayed in part by the payment of page class II transcription. charges. This article must therefore be hereby marked advertisement in accordance One mechanism often involved in regulating both the subcellu- with 18 U.S.C. Section 1734 solely to indicate this fact. lar localization and the activation of proteins is phosphorylation. 1 This work was supported by grants from the National Institutes of Health (AI29564, Phosphorylation of components of the nuclear transport machinery AI45580, and AI41751; to J.P.-Y.T.), a National Multiple Sclerosis Society Career Transition Award (to S.F.G.), an American Cancer Society fellowship (to J.A.H.), and globally down-regulates nuclear import, but does not affect nuclear a Milton J. Petrie-Irvington Institute fellowship and the National Science Foundation export (28). Additionally, phosphorylation of speciﬁc proteins, in- (MCB0212067; to D.E.C.). cluding cyclin B1 (29), NF-AT (30), and the yeast transcription 2 J.P.-Y.T. and D.E.C. contributed equally to this paper and should be considered co-senior authors. 3 Address correspondence and reprint requests to Dr. Drew E. Cressman, Department 4 Abbreviations used in this paper: GBD, GTP-binding domain; NLS, nuclear local- of Biology, Sarah Lawrence College, 1 Mead Way, Bronxville, NY 10708. E-mail ization signal; PKA, protein kinase A; RFX, regulatory factor X; TFIIB, transcription address: [email protected] factor class II B.

factor Pho4 (31), can alter their subcellular localization. We pre- dilution; BD Pharmingen, San Diego, CA), then rinsed and mounted in viously showed that CIITA is phosphorylated on a variety of dif- Vectashield mounting medium with DAPI (Vector Laboratories, Burlin- ϫ ferent serine residues, including a serine at aa 286 (32). Recently, game, CA), and observed at 50 magnification. additional phosphorylation sites have been assigned to the general Cell culture and luciferase assays region between aa 253–321 using deletion mutants. Although these HeLa human epitheloid carcinoma and COS-7 monkey kidney fibroblast specific mutants were not tested for transcriptional activity, a sep- cell lines were grown in DMEM supplemented with 10% FCS, 300 mg/L arate experiment in that paper showed that the treatment of cells L-glutamine, and 1% penicillin/streptomycin. Raji human lymphoma cells with inhibitors of phosphatase activity resulted in an increase in were grown in RPMI 1640 supplemented as described above. All cells were cultured at 37°Cin5%CO. All transfections were performed using CIITA trans-activation and that this is associated with an increase 2 FuGene 6 (Roche, Indianapolis, IN) according to the instructions of the in CIITA oligomerization and nuclear accumulation (33). Another manufacturer. Cells were split to six-well plates 12 h before transfection. paper mapped a protein kinase A (PKA) phosphorylation domain One microgram of each plasmid was transfected into cells along with 1 ␮g to the region between residues 325–408, with four serines identi- of the class II DR promoter fused to a luciferase reporter, and cells were ␮ ϫ fied as the PKA targets. Mutations of those four serines caused harvested at 24 h post-transfection in 200 lof1 reporter lysis buffer (Promega). Forty microliters were assayed for luciferase activity as previ- only a modest (2-fold or less) effect on the CIITA trans-activation ously described (36). The reporter plasmid for luciferase experiments con- function (34). Finally, a report showed that constitutively active tained ϳ300 bp of the DRA promoter (37) fused upstream of the luciferase protein kinase C increased CIITA expression, whereas a dominant gene in the pGL2-Basic vector (Promega). Extracts were normalized to negative mutant of protein kinase C blocked IFN-␥-induced MHC protein concentration, which was determined to be equivalent to normal- ␤ class II gene expression, although the phosphorylation sites were ization by cotransfected -galactosidase gene assays (data not shown). Each transfection was performed in triplicate. not mapped (35). Hence, there appears to be a complex array of phosphorylation targets. However, the functional consequence of Cellular fractionation and immunoblots Downloaded from phosphorylation on specific residues is less clear and represents a Cells were transfected in six-well plates with the indicated plasmids, har- focus of this report. vested at 24 h post-transfection, and fractionated into whole-cell, cytoplas- In this study we show that CIITA found in the nucleus, but not mic, or nuclear extracts. Whole-cell extracts were prepared by lysis of cells ␮ the cytoplasm, is predominantly phosphorylated, and we identify in 400 l of RIPA buffer (10 mM Tris (pH 7.4), 2% deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 50 mM NaF, and serines 286, 288, and 293 as phosphorylation targets. These find- 1ϫ protease inhibitors (Roche)). Cytoplasmic and nuclear extracts were ings largely agree with and extend the findings of the previous prepared as described previously (38). Protein concentrations of each frac- http://www.jimmunol.org/ report (36). Surprisingly, mutation of S293 together with S288 or tion were determined by the Bradford method. For immunoblot analyses, ␮ S286 significantly enhanced the nuclear translocation of CIITA 10 g of each fraction was separated on 5% SDS-polyacrylamide gels, transferred to nitrocellulose, and blotted with 2 ␮g of anti-FLAG M5 Ab and its ability to trans-activate the endogenous expression of MHC (Sigma-Aldrich). Anti-transcription factor class II B (TFIIB) Abs (Santa class II mRNA, indicating that whereas phosphorylation of these Cruz Biotechnology, Santa Cruz, CA) were used to blot for integrity of the residues is not required for nuclear import, it is important for me- extracts. For immunoprecipitation studies, whole-cell extracts were pre- diating the subsequent decrease in endogenous CIITA trans-acti- pared in RIPA buffer as described above. Extracts were incubated at 4°C ␮ ␮ vation ability. Together these findings suggest that CIITA function overnight with 2 g of anti-FLAG or anti-Myc Ab plus 20 l of Dyna- beads (Dynal Biotech, Great Neck, NY) per reaction. Beads were washed is down-regulated by phosphorylation at these critical residues. three times with RIPA buffer, boiled in 2ϫ loading buffer with SDS, and by guest on October 2, 2021 loaded onto 5% SDS-polyacrylamide gels. Materials and Methods Real-time PCR Plasmids and reagents cDNA was synthesized as described previously (39). Real-time PCR was All mutants were constructed using the pCDNA3.FLAG.CIITA parent vec- performed using the ABI PRISM 7900 sequence detection system tor containing an eight-amino acid FLAG epitope (DYKDDDDK) up- (PerkinElmer, Foster City, CA). MHC class II probes were labeled at the stream of the first methionine of CIITA (17). C-terminal CIITA deletion 5Ј end with the reporter dye FAM and at the 3Ј end with the quencher dye mutants were constructed by QuikChange site-directed mutagenesis (Strat- TAMRA. The 18S rRNA probe was labeled at the 5Ј end with the reporter agene, La Jolla, CA), resulting in a stop codon placed at the indicated dye TET and at the 3Ј end with the quencher dye TAMRA. Primer and position within the CIITA sequence. Stop mutations were made at codons probe sequences were previously reported (25), and real-time PCR analysis for aa 187, 225, 261, 271, 283, 294, 307, and 313. CIITA containing the ⌬ of cDNA products were conducted as previously described (39). Values internal deletion of aa 132–302 (CIITA 132–301) was constructed as de- ⌬ were calculated based on standard curves generated for each gene. Nor- scribed previously (17). CIITA 1–335 was made by introducing a BglII malization of samples was determined by dividing copies of MHC class II mutation at residue 334, then subcloning residues 335-1130 into BamHI/ by copies of 18S rRNA. EcoRI restriction sites in the pCDNA3-His C vector (Invitrogen, Carlsbad, CA). Point mutations of serine residues to alanine at aa 286, 288, and 293 Results were introduced by QuikChange site-directed mutagenesis. Double and triple mutants were generated by sequential mutagenic reactions. CI- Phosphorylated CIITA appears predominantly in the nucleus ITANLS and CIITA-5aa have been described previously (19). All con- During the course of previous studies of CIITA, we observed that structs were verified by sequence analysis. In vitro transcription/translation the protein migrates more slowly on SDS-polyacrylamide gels of wild-type CIITA was completed with TNT T7 Quick Master Mix (Pro- mega, Madison, WI) supplemented with 0.5 ␮g of plasmid DNA template than would be expected for an 1130-aa protein. FgCIITA migrates and 40 ␮M methionine. The reaction was incubated at 30°C for 60 min and as doublet through denaturing gels, with protein sizes of 149 and prepared for gel analysis. Where indicated, extracts were treated with 40 145 kDa as indicated on immunoblots (Fig. 1A, lane 1). We rea- ␮ ␭ U/ l protein phosphatase (New England Biolabs, Beverley, MA) at 30°C soned that one of these bands may be indicative of a phosphory- for1h. lated form of CIITA. When preincubated with a broad-acting ␭ Immunofluorescence protein phosphatase capable of dephosphorylating serine, threo- FLAG-tagged wild-type CIITA (1 ␮g) or the indicated CIITA mutants nine, and tyrosine residues, the upper 149-kDa band of CIITA were transfected into COS-7 cells growing in two-well chamber slides and completely disappears (compare lanes 1 and 2). Likewise, CIITA assayed for subcellular localization. Where indicated, 2.5 ␮g/ml actinomy- translated in vitro in the absence of any kinase also migrates only cin D (Sigma-Aldrich, St. Louis, MO) were added to the cells 3 h before as the lower band, 145-kDa protein (Fig. 1A, lane 3, and Fig. 1B, harvesting. At 24 h post-transfection, slides were rinsed in PBS, fixed in acetone/PBS (3/2 dilution) for 4 min, incubated 1 h with M5 anti-FLAG lane 7). Therefore, it appears that the upper, 149-kDa band repre- mouse mAb (Sigma-Aldrich) diluted 1/500 in PBS/1% BSA, rinsed, incu- sents the phosphorylated form of CIITA, whereas the lower, 145- bated 1 h with FITC-conjugated goat anti-mouse secondary Ab (1/500 kDa band represents the less phosphorylated form. It should be 378 PHOSPHORYLATION AND INHIBITION OF NUCLEAR CIITA

FIGURE 1. CIITA present in the nucleus is in a predominantly phosphorylated form. A, Immunoblot of extracts from COS cells transfected with wild- type CIITA (lanes 1 and 2), CIITA fused to an NLS at its C terminus (CIITANLS), which shows strong nuclear localization, or CIITA containing a deletion of the ﬁve amino acids comprising NLS2 ⌬ (CIITA 955–959), which is localized to the cytoplasm. Extracts from cells transfected with wild- type CIITA were initially treated with ␭ protein phosphatase (lane 2). In vitro transcribed and translated wild-type CIITA (i.v. CIITA) is shown in lane 3. All CIITA constructs contained an N-terminal FLAG epitope and were detected using an anti- FLAG Ab. B, Phosphatase treatment of CIITA elim- inates the larger form of the protein. COS cells were transfected with the indicated constructs, and ex-

tracts were incubated in the absence (–) or the pres- Downloaded from ence (ϩ)of␭ protein phosphatase before immunoblot analysis as in A. C, Immunoﬂuorescence of wild-type (CIITA), nuclear (CIITANLS), and cytoplasmic (CIITAD955–959) forms of CIITA. Expres- sion plasmids were transfected into COS cells. Flu- orescence indicates the subcellular localization of

the protein. D, Immunoblot of subcellular fraction- http://www.jimmunol.org/ ations of COS cells transfected with wild-type CIITA. Cytoplasmic and nuclear fractions were separated by electrophoresis, and the doublet pattern of CIITA was detected with an anti-FLAG Ab. The integrity of the extracts was conﬁrmed by immuno- blotting for the NF TFIIB in both fractions. Com- mercially prepared, untransfected, HeLa whole-cell extract was used as a control for TFIIB protein. by guest on October 2, 2021

noted that despite treatment with the phosphatase or in vitro trans- sively the 145-kDa, and thus unphosphorylated, form of CIITA, lation, the lower band does not decrease in size, suggesting that whereas both unphosphorylated and the more abundant 149-kDa this is the minimal molecular mass of unphosphorylated, full- phosphorylated CIITA are present in nuclear extracts (Fig. 1D). length CIITA (Fig. 1B). We have previously observed that CIITA The integrity of the extracts was confirmed by blotting for the is present in both the cytoplasm and the nucleus and that mutating nuclear basal transcription factor TFIIB and comparing to the the CIITA gene can alter this differential subcellular localization known size of TFIIB derived from commercially prepared HeLa (19, 20). Addition of a C-terminal NLS to CIITA (CIITANLS) cell extracts. increases the presence of CIITA in the nucleus, whereas deletion of a five-amino acid NLS motif (residues 955–959) missing Identification of potential sites of phosphorylation on CIITA in the CIITA gene of bare lymphocyte syndrome patients To identify sites of phosphorylation within CIITA, various N-ter- ⌬ (CIITA 955–959) causes retention of the protein in the cytoplasm minal deletions were made and assayed for loss of the upper, phos- (Fig. 1C). Upon immunoblot examination, we found that cells phorylated form of CIITA in extracts of transfected cells (Fig. 2). transfected with CIITANLS showed an increase in the presence of Deletion of aa 1–335 resulted in a single, unphosphorylated protein phospho-CIITA relative to wild-type (Fig. 1A, lane 4), whereas band detected on immunoblots (Fig. 2A), suggesting that phos- ⌬ cells transfected with CIITA 955–959 exhibited a marked decrease phorylation occurs in aa 1–335. An internal deletion of aa 132–301 in the amount of phosphorylated protein present in extracts (lane also resulted in a single, unphosphorylated CIITA band, further 5). These results were confirmed by treatment of each of these delineating the region containing possible sites of phosphorylation. CIITA constructs with ␭ phosphatase, which eliminated only the Deletion of increasing amounts of the C terminus revealed that upper band while leaving untouched the lower band, representing CIITA constructs of residues 1–186, 1–224, and 1–260 all re- the smaller form of CIITA (Fig. 1B). This suggests that CIITA mained as single unphosphorylated forms (Fig. 2B). In contrast, present in the nucleus is predominantly phosphorylated, whereas CI- CIITA 1–306 appears as a double band, and loss of the upper, ITA remaining in the cytoplasm is only minimally phosphorylated. phosphorylated band is evident after treatment with phosphatase. To confirm that CIITA is differentially phosphorylated depend- Further mapping of potential phosphorylation sites by immuno- ing on its subcellular localization, we prepared nuclear and cyto- blots of CIITA deletion mutants suggested that phosphorylation plasmic extracts from cells transfected with wild-type CIITA. leading to the appearance of a CIITA doublet occurs between res- Cytoplasmic fractions of transfected cells contain almost exclu- idues 282 and 293 (Fig. 2C). Phosphoamino acid peptide analysis The Journal of Immunology 379

tively), whereas mutation of serine 288 alone (S288A) resulted in a relative decrease in the upper, phosphorylated form of CIITA (lane 3). Combined S286/288A or S288/293A mutations have the most pronounced effect on formation of the CIITA doublet, resulting in a marked decrease in the presence of phosphorylated CIITA (lanes 5 and 7). Interestingly, the S286/293A double mutant showed increased levels of phosphorylation. Potentially, this mutant causes an alteration in the CIITA structure that permits phosphorylation of other residues in more distant regions of the protein, although this possibility remains to be investigated. Mutation of all three serine residues together resulted in a relatively unstable CIITA protein expressed at a lower efficiency. Therefore, we focused on single and double serine mutants in the course of exam- ining their effects on phosphorylation and localization. Mutation of serine residues induces nuclear localization of CIITA As immunoblot analyses indicate that the nuclear form of CIITA appears to be heavily phosphorylated, whereas the less phosphorylated CIITA form resides in the cytoplasm, we determined Downloaded from whether mutation of the serine residues alters CIITA localization. COS cells were transfected with wild-type CIITA or CIITA mutated at serines 286, 288, and 293 (S286A, S288A, and S293A, respectively). Immunofluorescence of these transfected cells showed that wild-type CIITA is normally present in both the cytoplasm and the nucleus (Fig. 3), agreeing with previous studies http://www.jimmunol.org/ (19, 20). As single-point mutations showed a slight increase in the nuclear concentration of CIITA, double-point mutations (S286/ 288A, S288/293A, and S286/293A) were also examined for their effects on CIITA localization. Each of the CIITA double mutants showed an increase in nuclear concentration of CIITA, with the mutants that include the serine-to-alanine mutation at residue 288 (S286/288A and S288/293A) showing a significant increase in nu- FIGURE 2. Deletion mapping identifies phosphorylated serine residues clear concentration. Thus, whereas mutation of the serine residues in CIITA. Immunoblot analyses of extracts from COS cells transfected by guest on October 2, 2021 with the indicated FLAG-tagged wild-type or mutant CIITA genes. A, The alone only mildly increases CIITA in the nucleus, double muta- doublet CIITA bands are present in wild-type CIITA-transfected cells, but tions, especially mutation of serine 288 in conjunction with a mu- absent in extracts from cells transfected with CIITA missing the N-terminal tation at either of the other two serine sites (S286 or S293), are ⌬ 335 aa ( 1–335) or CIITA containing an internal deletion of residues 132– sufficient to enhance CIITA’s presence in the nucleus. If serine ⌬ 301 ( 132–301). B, Detailed mapping of the region of CIITA between res- phosphorylation is responsible for translocation of CIITA from the idues 186 and 306. Cells were transfected with C-terminal deletion mutants cytoplasm into the nucleus, then it would be expected for the mu- of CIITA, such that the mutant CIITA proteins contained only the indicated tants to demonstrate increased cytoplasmic localization. However, ␭ amino acids. Ten micrograms from each extract was incubated with the opposite was observed, indicating that phosphorylation of these protein phosphatase and compared with untreated extracts after SDS- sites is not required for nuclear import. Instead, phosphorylation at PAGE separation and immunoblot. Note the loss of the upper band after these residues may act as a subsequent signal to target CIITA for phosphatase treatment of extracts from cells transfected with CIITA1–306. C, Fine mapping of the region between aa 260 and 306. Loss of the upper nuclear export and/or proteasomal degradation and thereby alter CIITA function. band detected with the anti-FLAG Ab in CIITA1–293 coincides with deletion of the adjacent 11 aa generating CIITA1–282. D, Banding patterns Effect of phosphorylation on CIITA self-association or from extracts of cells transfected with CIITA constructs mutated at the three serine residues between aa 282 and 293. Serine-to-alanine mutations interaction with other proteins were made individually at serine 286, 288, or 293 (lanes 2Ð4) and in If phosphorylation is necessary to modulate nuclear CIITA func- combinations in which two serine residues were mutated together (lanes tion, we explored the possibility that phosphorylation specifically 5Ð7). The ratio of the upper, phosphorylated band to the lower, unphos- alters the critical interactions of CIITA with itself or other factors. phorylated band in each lane is indicated at the bottom. The SD is indicated CIITA undergoes self-association, mediated by several domains from three independent experiments. E, Amino acid sequence of the region spread throughout the N terminus, the nucleotide binding domain between aa 282 and 312 of CIITA. Potential phosphorylated residues iden- tified above are boxed. and leucine-rich region (21, 33, 34, 40). In addition, nuclear CIITA interacts with transcription factors such as RFX5 and NF-Y in trimeric complexes at the promoters of MHC class II target genes suggests that CIITA is phosphorylated exclusively on serine resi- to activate transcription. To determine whether the phosphorylated dues (32), and within this region of CIITA are three serine residues or unphosphorylated form of CIITA self-associates or interacts at aa 286, 288, and 293 (Fig. 2E). We analyzed these particular with transcription factors to form the MHC class II enhanceosome, residues by mutating each of these sites alone or in combination we performed coimmunoprecipitations of CIITA from extracts of with one another (Fig. 2D). Mutation to alanine of serine 286 transfected cells. Cells were transfected with FLAG-tagged CIITA (S286A) and serine 293 (S293A) did not result in a dramatic (FgCIITA) and CIITA, RFX5, or NF-YB tagged with the Myc change in the proportion of the two forms (lanes 2 and 4, respec- epitope. Extracts were immunoprecipitated with anti-FLAG or anti- 380 PHOSPHORYLATION AND INHIBITION OF NUCLEAR CIITA Downloaded from http://www.jimmunol.org/ FIGURE 4. Interaction of CIITA with itself or other proteins is not dependent on phosphorylation of CIITA. A, Extracts from cells transfected with FLAG-tagged CIITA (FgCIITA) and cotransfected with Myc-tagged CIITA, RFX5, or NF-YB were subjected to immunoprecipitation with anti- FLAG or anti-Myc Abs or were incubated with anti-Ig beads alone (Ϫ). FLAG-tagged immunoprecipitated proteins were detected by immunoblot- ting with the anti-FLAG Ab. Note the presence of both phosphorylated and unphosphorylated FgCIITA coimmunoprecipitating with the MycCIITA, FIGURE 3. Double-point mutations that include serine 288 increase the MycRFX5, and MycNF-YB. B, Dephosphorylation does not eliminate self- nuclear localization of CIITA. COS cells were transfected with the indi- association of CIITA. Extracts from FgCIITA- and MycCIITA-transfected by guest on October 2, 2021 cated FLAG-tagged constructs, and immunofluorescence was performed cells were subjected to phosphatase treatment, then immunoprecipitated as 24 h post-transfection using an anti-FLAG Ab to detect the subcellular described in A. localization of CIITA. All cells were treated with actinomycin D for 3 h before harvest to ensure that any cytoplasmic protein was not simply the result of new synthesis. Samples were compared with the relatively even cellular distribution of wild-type CIITA. tracts immunoprecipitated with anti-Myc Ab were able to coim- munoprecipitate FLAG-tagged CIITA, indicating that FgCIITA and MycCIITA remained associated despite treatment with phos- Myc Abs, and complexes were detected by immunoblot with the phatase. Taken together, the data indicate that in the presence of anti-FLAG Ab (Fig. 4A). In each set, the control immunoprecipi- both forms of the protein, there was a preferential association be- tation with anti-FLAG detected both phosphorylated and unphos- tween the phosphorylated forms. However, removal of the phosphorylated CIITA, indicating the presence of both species within phate groups did not disrupt homodimerization. There can be at the cell. Immunoprecipitations with anti-Myc Ab, followed by im- least two interpretations: one is that dephosphorylated CIITA is the munoblot with anti-FLAG to detect CIITA-associated CIITA or less preferred form for self-association, but in the absence of phos- RFX5, predominantly detected the higher molecular mass phosphorylated CIITA, its self-associative property becomes more ev- phorylated form of CIITA, suggesting that the phosphorylated ident. Alternatively, it is possible that phosphorylation is required form may preferentially associate with CIITA and RFX5 (Fig. 4A, for self-association, but not for the maintenance of self-associa- lanes 1Ð6). A similar experiment performed with NF-Y also de- tion. Hence, once CIITA self-associates in cells, the removal of tected the higher molecular mass phosphorylated form of CIITA in phosphates has no effect on self-associated dimers or multimers. association with NF-Y. The preferential association of CIITA and RFX5 with a phosphorylated form of CIITA is in agreement with Double mutation of serine residues has a small effect on CIITA previous publications (33, 34). trans-activation of an artificial promoter-reporter system As CIITA preferentially immunoprecipitated the phosphorylated We next examined the effect of phosphorylation on CIITA-medi- form of itself, self-association of CIITA appears to be dependent on ated gene activation. COS and HeLa cells were cotransfected with the state of protein phosphorylation. To confirm this result, extracts wild-type CIITA or the serine mutants along with a luciferase re- from cells transfected with FgCIITA and MycCIITA were incu- porter under the control of the MHC class II HLA-DR promoter. bated with the ␭ protein phosphatase before immunoprecipitation CIITA activated expression from the DR promoter by 25- to 40- (Fig. 4B). In the absence of protein phosphatase, the phosphory- fold in both cell lines. Single-point mutants have minimal impact lated band of CIITA was preferentially coprecipitated, as observed on altering activation of the MHC class II promoter (Fig. 5A). in Fig. 4A. However, despite treatment with the phosphatase, ex- Double-serine mutants, S286/288A and S288/293A, both modestly The Journal of Immunology 381

FIGURE 6. CIITA phospho-mutants increase the expression of endogenous MHC class II genes. Real-time PCR analysis was performed to mea- sure endogenous mRNA levels of class II in HeLa cells transfected with CIITA phosphorylation mutants. Eighteen hours after cell culture, HeLa

cells were transfected with 500 ng of pCDNA3 (control), wild-type CIITA, Downloaded from or CIITA double mutants, and mRNA was isolated 24 h post-transfection. Samples were normalized to the number of 18S rRNA copies. The data shown are representative of three experiments.

with wild-type CIITA. Therefore, loss of phosphorylation within the region of S286-S293 of CIITA leads to increased activation of http://www.jimmunol.org/ endogenous MHC class II genes, suggesting that phosphorylation of CIITA at the indicated amino acids served to down-regulate the normal protein activity.

FIGURE 5. Mutation of serine 288 in double-serine mutants enhances Discussion the trans-activation ability of CIITA. Serine-to-alanine mutations at resi- CIITA has been well characterized as a transcriptional activator dues 286, 288, and 293 were assayed singly (A) or in each double com- operating at the promoter of MHC class II target genes. Recent f ^ bination (B). COS ( ) or HeLa cells ( ) were transfected with wild-type evidence suggests that CIITA is present in the cytoplasm as well by guest on October 2, 2021 CIITA or the indicated CIITA mutants along with the luciferase reporter (18, 19) due to export of the protein from the nucleus (20, 27). The under control of the class II DR promoter. One hundred percent activity in question arises of how this dual presence is controlled and what each cell type was defined as the level of DR-luciferase activation by wild- type CIITA. All other values were plotted as percentages of this activity. modifications of CIITA may regulate its localization and activity. All experiments were performed in triplicate and normalized to protein One mechanism commonly used by cells to control protein func- concentration. tion is that of phosphorylation. Numerous kinase pathways are activated within the cell after the stimulation of cell surface re- ceptors with a variety of cytokines or other factors. For example, enhanced activation by 30–60% more than that seen for wild-type the expression of MHC class II genes is stimulated by IFN-␥ via CIITA, whereas the S286/293A mutant showed activity levels activation of the Janus kinases through Stat1 and IFN regulatory comparable to those of wild-type CIITA (Fig. 5B). These levels are factor-1 activation of CIITA expression (1, 41, 42). The function too modest, particularly for a promoter-reporter system, to permit or localization of a variety of other proteins has been shown to be us to reach a conclusion about the effects of these serine mutations modulated by phosphorylation (43). In this paper we have at- on CIITA activity. tempted to further define the roles of specific serine residues as targets of phosphorylation and as residues important for modulat- Double mutation of serine residues significantly enhanced CIITA ing CIITA localization and activity. trans-activation of an endogenous MHC class II gene Subcellular fractionations and immunoblot assays indicated that The transfected DR-luciferase reporter construct used above rep- nuclear CIITA is predominantly phosphorylated, although a resents an artificial system without the benefit of fully assembled smaller amount of unphosphorylated CIITA is still evident in nu- chromatin structures at the tested promoter. To thoroughly assess clear fractions. In contrast, CIITA in the cytoplasm is primarily the impact of phosphorylation on CIITA activity, it was important unphosphorylated, suggesting that either phosphorylated CIITA is to establish the effects of CIITA phosphorylation on regulation of immediately transported from the cytoplasm into the nucleus or the expression of endogenous MHC class II genes. Cells trans- that after import into the nucleus, unphosphorylated CIITA is tar- fected with wild-type CIITA or double-point mutants were exam- geted by a nuclear kinase. In vitro phosphopeptide-mapping stud- ined for levels of endogenous MHC class II gene expression, as ies have indicated multiple sites of serine phosphorylation on measured by quantitative real-time PCR analysis (Fig. 6). The ex- CIITA (32). Through analysis of deletion mutants, we have nar- pression of MHC class II genes induced by double mutants of rowed the region down to three serine residues, aa 286, 288, and CIITA was compared with that induced by wild-type CIITA ex- 293, which serve as primary targets of phosphorylation. Deletion pression. Surprisingly, cells transfected with S288/293A and S286/ of a region containing these residues results in a form of CIITA 293A mutants consistently showed a 3- to 5-fold increase in levels that is unaffected by treatment with the ␭ protein phosphatase. of MHC class II gene expression over that seen in cells transfected Site-specific mutation of any two of these sites caused varying 382 PHOSPHORYLATION AND INHIBITION OF NUCLEAR CIITA

‘degrees of altered phosphorylation; however, some phosphory- tion within the region of residues 286–293 may play a role in lated CIITA remained. Mutation of all three resulted in very low down-regulating CIITA activity. The increased trans-activation protein expression, probably attributed to protein instability, such ability of these CIITA phospho-mutants may be directly related to that the results are inconclusive (not shown). Although these ex- their increased concentration within the nucleus, as is apparent in periments indicate that these three serine residues probably en- immunoﬂuorescence assays. Therefore, the role of phosphoryla- compass major phosphorylation sites, recent studies have sug- tion at these residues may be to decrease the nuclear concentration gested additional regions between the proline-serine-threonine and of CIITA. As suggested previously, this may be achieved by en- GBD of CIITA as targets of PKA-mediated phosphorylation (34). hancing protein degradation or nuclear export. A direct conse- Another paper showed that the region 253–321 contains the phos- quence of this decrease in nuclear CIITA is the down-regulation of phorylation sites, which agrees with our mapping of serines 286, CIITA-mediated gene expression, as evidenced by the increased 288, and 293 as the most probable sites, although additional levels of endogenous MHC class II message seen in the presence serines within that region may contribute to global CIITA phos- of CIITA phosphorylation mutants. phorylation (33). There are several explanations to interpret these data in the light The possible roles of phosphorylation of CIITA include regu- of the earlier data. Data presented in this report and in two earlier lating nuclear transport, protein-protein interactions, or gene acti- reports indicate that the phosphorylated form of CIITA enhances vation. The phosphorylated form is more prominent in the nucleus, self-association and interaction with RFX5 and NF-Y (33, 34). whereas the nonphosphorylated form is more prominent in the cy- Those studies also implicate a positive effect of kinases and a neg- toplasm. This was shown in a number of experiments, including ative effect of phosphatases on CIITA function. In contrast, we cell fractionation and the test of mutants that primarily localize to

demonstrate that speciﬁc mutation of serines 288 and 293 de- Downloaded from the cytoplasm or nucleus. This indicates that the coupling of phos- creases the amount of the phosphorylated form of CIITA and in- phorylation and nuclear import may occur very rapidly, such that creases CIITA-mediated endogenous gene expression. It is impor- little of the phosphorylated cytoplasmic form is ever detected, or tant to note that Tosi et al. (33) did not test CIITA mutants that that CIITA is primarily phosphorylated in the nucleus. We favor lacked the region of phosphorylation in a trans-activation assay, the latter explanation, as mutations of these sites did not block but inferred the importance of phosphorylation as a positive reg- nuclear translocation and actually increased nuclear localization of

ulatory step by the use of phosphatase or phosphatase inhibitors. In http://www.jimmunol.org/ the protein. This increase in the nuclear concentration of the experiments in which they transfected recombinant CIITA into phosho-mutant form of CIITA suggests that phosphorylation U937 cells, the protein was predominantly nonphosphorylated, and within the region of aa 286–293 occurs within the nucleus and little MHC class II was detected, unless PMA was added, in which serves to decrease nuclear CIITA in one of two ways: it may fa- case CIITA was phosphorylated and MHC class II expression was cilitate export of CIITA back to the cytoplasm, or it may induce increased. However, CIITA mutants again were not tested (33). degradation of the phosphorylated CIITA protein in the nucleus. Sisk et al. (34) focused on in vitro phosphorylation of CIITA by Therefore, we suggest that the ability of CIITA to enter the nucleus PKA to demonstrate that maximal CIITA activity requires PKA is independent of the state of phosphorylation of residues 286– phosphorylation in the region of CIITA aa residues 325–408. This 293. Subsequent phosphorylation of serines within this region af- constitutes a different issue and concerns PKA-induced phosphor- by guest on October 2, 2021 fects nuclear CIITA by inducing a decrease in the total nuclear ylation, which is known to either enhance or reduce MHC class II concentration of the protein. depending on the cell type. A specific mutant bearing mutations in With regard to the roles of phosphorylation and protein interaction, both underphosphorylated and phosphorylated CIITA can all four serine residues within the target region showed decreased interact with RFX5 and NF-YB, although the phosphorylated form activity compared with wild-type CIITA; however, the effect was interacts better with RFX5. Data for NF-YB show a similar trend, modest, and only a reporter system was used. Each study used but the evidence was less conclusive. Self-association of CIITA also different approaches to identify a role for phosphorylation in reg- appears to prefer the phosphorylated form, but dephosphorylation by ulating CIITA activity. The most likely conclusion is that a com- ␭ protein phosphatase does not eliminate CIITA self-interactions. plex pattern of phosphorylation by different kinases on different These observations indicate either that dephosphorylation does not residues of CIITA in response to different stimuli may result in disrupt preformed CIITA dimers/multimers, or that in the absence of divergent outcomes. Phosphorylation can lead to enhanced CIITA phosphorylated CIITA, which serves as a superior partner, dephos- self-association and association with other DNA-binding factors phorylated CIITA can undergo self-association. and probably enhanced transcription. In contrast, the specific re- The enhanced association of phosphorylated CIITA with itself gion of residues 286–293 has a negative regulatory function. One and with RFX5 led us to initially hypothesize that phosphorylation caveat is that the functional effect of mutating residues 293 in of CIITA at S286, S288, and S293 is probably a positive regula- combination with 286 or 288 may be unrelated to phosphorylation tory step, similar to that proposed by others (33, 34). Our initial and simply due to a change of amino acid side chains. We cannot trials with mutants of S286/288A and S288/293A showed a small rule out this possibility at present, although this region clearly increase in trans-activation ability in promoter reporter assays; contains the predominant phosphorylation sites, as removal of this however, the changes were very slight, such that conclusions could region reduced the phosphorylated form. not be drawn. Surprisingly, further analysis of the effects of CIITA The data presented in this paper suggest the following model for double mutations on endogenous MHC class II gene expression by the role of phosphorylation of aa S286, S288, and S293 on CIITA real-time PCR indicated that double mutants that included serine activity. Cytoplasmic CIITA is relatively underphosphorylated. 293 showed significantly enhanced CIITA activity. The difference Phosphorylation of CIITA probably occurs in the nucleus or con- in MHC class II gene expression levels between the promoter- currently with translocation of the protein into the nucleus, as the reporter luciferase assay and the real-time PCR experiments may phosphorylated form of CIITA is found predominantly associated be due to the fact that only the latter measured CIITA-mediated with nuclear fractions. CIITA may be phosphorylated at sites other gene expression from intact chromatin and was therefore a more than S286, S288, and S293 to cause preferential association with physiological assessment of promoter activation. Thus, phosphor- RFX5 and other transcription factors to activate the MHC class II ylation of individual serines 288 and 293 as well as phosphoryla- target genes. As a molecular switch, specific phosphorylation of The Journal of Immunology 383

CIITA residues S286, S288, and S293 later causes down-regula- 19. Cressman, D. E., K. C. Chin, D. J. Taxman, and J. P. Ting. 1999. A defect in the tion of its trans-activation ability. This may lead to enhanced deg- nuclear translocation of CIITA causes a form of type II bare lymphocyte syndrome. Immunity 10:163. radation of CIITA or increased export to reduce the nuclear local- 20. Cressman, D. E., W. J. O’Connor, S. F. Greer, X. S. Zhu, and J. P. Ting. 2001. ization of CIITA and its trans-activation function, both of which Mechanisms of nuclear import and export that control the subcellular localization of class II transactivator. J. Immunol. 167:3626. can be tested. Furthermore, CIITA with mutated S286, S288, and 21. Kretsovali, A., C. Spilianakis, A. Dimakopoulos, T. Makatounakis, and S293 should not exhibit reduced ability to associate with RFX and J. Papamatheakis. 2001. Self-association of class II transactivator correlates with other transcription factors and may even exhibit enhanced asso- its intracellular localization and transactivation. J. Biol. Chem. 276:32191. 22. Spilianakis, C., J. Papamatheakis, and A. Kretsovali. 2000. Acetylation by PCAF ciative capacity, correlating with its enhanced ability to activate enhances CIITA nuclear accumulation and transactivation of major histocompat- the endogenous MHC class II gene. Various aspects of this model ibility complex class II genes. Mol. Cell. Biol. 20:8489. are testable, and future experiments should allow us to better de- 23. Harton, J. A., E. Zika, and J. P. Ting. 2001. The histone acetyltransferase domains of CBP and pCAF are not necessary for cooperativity with CIITA. J. Biol. Chem. cipher the relevance of these three serine residues as well as other 20:20. phosphorylated residues in modifying the function of CIITA. 24. Greer, S. F., E. Zika, B. Conti, X. S. Zhu, and J. P. Ting. 2003. 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