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.     

CREB-binding protein/p300 activates MyoD by acetylation

The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells. In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo.     

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.