Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator

Abstract

The expression of major histocompatibility class II genes is necessary for proper antigen presentation and induction of an immune response. This expression is initiated by the class II transactivator, CIITA. The establishment of the active form of CIITA is controlled by a series of post-translational events, including GTP binding, ubiquitination, and dimerization. However, the role of phosphorylation is less clearly defined as are the consequences of phosphorylation on CIITA activity and the identity of the kinases involved. In this study we show that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) interact directly with CIITA, targeting serine residues in the amino terminus of the protein, including serine 288. Inhibition of this phosphorylation by dominant-negative forms of ERK or by treatment of cells with the ERK inhibitor PD98059 resulted in the increase in CIITA-mediated gene expression from a class II promoter, enhanced the nuclear concentration of CIITA, and impaired its ability to bind to the nuclear export factor, CRM1. In contrast, inhibition of ERK1/2 activity had little effect on serine-to-alanine mutant forms of CIITA. These data suggest a model whereby ERK1/2-mediated phosphorylation of CIITA down-regulates CIITA activity by priming it for nuclear export, thus providing a means for cells to tightly regulate the extent of antigen presentation.

FIGURE 1.

ERK2 and Cdc2 directly phosphorylate CIITA in vitro. A, in vitro transcribed and translated FgCIITA was incubated alone (second lane) or with the indicated serine/threonine kinases followed by immunoblot analysis with an anti-Fg antibody. The presence of single or double bands was compared with doublets of the FgCIITA protein present in whole cell extracts prepared from COS7 cell lysates (first lane). CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; CKII, casein kinase II. B, incubation of FgCIITA phosphorylated in vitro by ERK2 with λ-protein phosphatase eliminates the upper band of the doublet. Whole cell extracts (WCE) from FgCIITA transfected cells or in vitro prepared FgCIITA were incubated with ERK2 and λ-protein phosphatase as indicated and detected as in A. Single (C) or double point mutants (D) of CIITA at serine 286 (S286A), serine 288 (S288A), or serine 293 (S293A) were subjected to in vitro transcription and translation followed by exposure to ERK2 and immunoblot detection by anti-Fg.

FIGURE 2.

CIITA interacts with exogenous and endogenous ERK1/2. A, co-immunoprecipitation of CIITA·ERK1/2 complexes in COS7 cells transfected with Xp-tagged CIITA, CMV-ERK1, and Fg-tagged ERK2. After preparation of whole cell extracts, immunoprecipitations (IP) were performed using anti-tubulin, anti-ERK2, or anti-Xp antibodies, followed by anti-ERK2 or anti-Xp immunoblotting (IB) as indicated. Immunoblots of whole cell extracts (WCE) were performed as controls for the level of XpCIITA or ERK1/2 expression. B, endogenous ERK1/2 interacts with CIITA. Co-immunoprecipitation of CIITA·ERK1/2 complexes in COS7 cells transfected with Fg-tagged CIITA alone followed by immunoblots with anti-Fg or anti-ERK of immunoprecipitated complexes or whole cells extracts as above. Immunoprecipitation with the anti-tubulin antibody serves as a negative control.

FIGURE 3.

CIITA activity increases in the presence of dominant-negative ERK1/2 and a MAPK inhibitor. COS7 cells were transfected with wild-type CIITA along with a luciferase reporter gene under the control of the MHC class II DR promoter. Indicated samples were also co-transfected with ERK1/2 or dominant-negative ERK1/2 or treated with U0126 and PD98059 at the time of transfection. 24 h post-transfection extracts from cells were prepared and assessed for luciferase activity. 100% activity in 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. All experiments were performed in triplicate and normalized to protein concentration. ^*, p < .05 compared with CIITA alone.

FIGURE 4.

Inhibition of endogenous ERK1/2 by dominant-negative mutants or PD98059 increases the nuclear localization of CIITA. COS7 cells were transfected with either wild-type XpCIITA (A-D) or the S288A/S293A double serine mutant (E-H) and co-transfected with ERK1/2 or dominant-negative ERK1/2 or treated with PD98059 at the time of transfection. Immunofluorescence was performed 24 h post-transfection using anti-Xp to detect the subcellular localization of CIITA. Samples were compared with the relatively even nuclear-cytoplasmic distribution of wild-type CIITA (A). DAPI, 4′,6-diamidino-2-phenylindole.

FIGURE 5.

Interaction between CIITA and CRM1 is decreased after inhibition of ERK1/2. Shown is co-immunoprecipitation (IP) of COS7 cells transfected with FgCIITA and co-transfected with dominant-negative ERK1/2 or treated with PD98059 as indicated. Extracts from cells were made 24 h post-transfection or treatment, then were subject to immunoprecipitation using anti-Fg (first through third lanes) or anti-CRM1 (fourth through sixth lanes) antibodies. FgCIITA present in the immunoprecipitated complexes was detected by immunoblotting (IB) with anti-Fg antibodies. Arrows indicate bands showing phosphorylated (upper) and unphosphorylated (lower) forms of CIITA. Immunoblots of whole cell extracts (WCE) were performed as controls for the level of FgCIITA expression.

FIGURE 6.

Inhibition of ERK1/2 increases endogenous CIITA activity and synergistically activates CIITA in a macrophage cell line. Raw 264.7 macrophage cells were transfected with the luciferase reporter under the control of the MHC class II DR promoter. Indicated cells were co-transfected with dnERK1/2 (lanes 2, 5), exogenous FgCIITA (lanes 4-6), or were treated with PD98059 (lanes 3, 6). Extracts were prepared at 24 h post-transfection and assayed for luciferase activity. The level of DR-luciferase activation by endogenous CIITA alone (first lane) was defined as 100% activity, and values of the remaining samples were plotted as percentages relative to this level. All experiments were performed in triplicate and normalized to protein concentration.