Identification of a nuclear export sequence in the MHC CIITA.

Initiation of an immune response through expression of MHC class II and related genes is under the control of the CIITA. Normally found in both the cytoplasm and nucleus, CIITA is tightly controlled by a variety of posttranslational modifications as well as interactions with other nuclear and cytoplasmic factors, whereas disruption of this dual subcellular localization impairs CIITA functioning and expression of target genes. Although CIITA has well-defined domains necessary for its nuclear import, the region responsible for the translocation of CIITA from the nucleus has not been characterized. In this study, we identify a leucine-rich motif at residues 717-724 that bears strong homology to known nuclear export sequence (NES) domains. Mutation of this region renders CIITA insensitive to treatment with leptomycin B, an inhibitor of nuclear export, whereas fusion of this domain to a heterologous GFP is sufficient to induce its export to the cytoplasm or cause its retention in the nucleus following leptomycin B treatment. Point mutations of specific leucine residues within the NES disrupt the normal subcellular distribution of the full-length CIITA, impair its ability to interact with the nuclear export factor CRM1, and enhance CIITA-induced gene expression from an MHC class II gene promoter. IFN-γ stimulation of class II genes is further enhanced by inhibiting the nuclear export of endogenous CIITA. Collectively, these data demonstrate the first identification of a specific NES within CIITA and place it among the other protein domains that contribute to the posttranslational regulation of CIITA activity.

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

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.

Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation.

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 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.