The invariant chain is a phosphorylated subunit of class II molecules

The phosphorylation of the MHC, class II-associated invariant chain (gamma) is demonstrated in human B-lymphoblastoid, melanoma, and histiocytic lymphoma cell lines. Two-dimensional nonequilibrium gel electrophoresis of invariant chain and class II Ag immunoprecipitates isolated from [32P]orthophosphate-labeled cells demonstrates labeling of both free and class II-associated gamma, gamma s, and p41 forms of the invariant chain. The gamma 2/gamma 3 form of the invariant chain is not phosphorylated. Phosphoamino amino acid analysis of isolated invariant chain shows phosphorylation of serine residues. The isolation of invariant chain from 32P-labeled microsome preparations digested with proteinase K demonstrates that the phosphorylation occurs in the cytoplasmic tail. Limited proteolysis of [32P]orthophosphate-, [35S]cysteine-, and [35S]methionine-labeled invariant chain also indicates that the 32P-label is incorporated into the cytoplasmic domain. These results pinpoint serine residues at positions 9, 26, and 29 in the N-terminal cytoplasmic tail as potential sites for the phosphorylation of the invariant chain. Phosphorylation may be another mechanism by which the functions of invariant chain in class II-dependent immune responses are regulated.     

Human major histocompatibility complex class II invariant chain is expressed on the cell surface.

Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.     

Regulation of MHC class II invariant chain expression: induction of synthesis in human and murine plasmocytoma cells by arresting replication.

The expression of the H2 Ia-associated invariant chain (Ii) has been determined by pulse labeling cells with [35S]methionine and resolving the proteins. Expression is maximal in noncycling peripheral B lymphocytes and is reduced upon maturation of B lymphocytes to plasma cells. B cell-derived cell lines behave correspondingly: IgM+ non-secretor cell lines synthesize Ii while plasmocytoma cells do not. Pre-B cell lines are also negative. In all Ii-negative cell lines, the synthesis of Ii is selectively induced by treating the cells with inhibitors of replication.     

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.     

Regulation of MHC class II gene expression by the class II transactivator

MHC class II molecules are pivotal for the adaptive immune system, because they guide the development and activation of CD4+ T helper cells. Fulfilling these functions requires that the genes encoding MHC class II molecules are transcribed according to a strict cell-type-specific and quantitatively modulated pattern. This complex gene-expression profile is controlled almost exclusively by a single master regulatory factor, which is known as the class II transactivator. As we discuss here, differential activation of the three independent promoters that drive expression of the gene encoding the class II transactivator ultimately determines the exquisitely regulated pattern of MHC class II gene expression.     

Interaction of MHC class II molecules with the invariant chain: role of the invariant chain (81-90) region.

Association of the invariant chain (Ii) with MHC class II alpha and beta chains is central for their functionality and involves the Ii CLIP(81-104) region. Ii mutants with an antigenic peptide sequence in place of the CLIP region are shown to form alphabetaIi complexes resistant to dissociation by SDS at 25 degrees C. This reflects class II peptide binding site occupancy, since substitution of the major anchor residue within the antigenic peptide sequence of one of these Ii mutants abolishes its capacity to form SDS-stable heterotrimers. Therefore, CLIP location within Ii is compatible with CLIP access to the class II binding groove. However, in wild-type Ii this access does not lead to a tight association, which seems to be affected by the Ii 81-90 region. This region, together with a region C-terminal of CLIP, is shown to contribute to Ii association with HLA-DR1 molecules. Thus, Ii mutants with non-HLA-DR1 binding sequences in place of the CLIP(87-102) region can still associate with HLA-DR1 molecules and inhibit peptide binding.     

A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein.

A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene.     

Phosphorylation of the invariant chain by protein kinase C regulates MHC class II trafficking to antigen-processing compartments.

The invariant chain (Ii) plays a critical role in the transport of newly synthesized class II molecules to endosomal Ag-processing compartments. Of the two major isoforms of human Ii, only Ii-p35 is phosphorylated in vivo, and inhibiting Ii phosphorylation inhibits the trafficking of newly synthesized class II molecules to Ag-processing compartments. We now report that a member of the protein kinase C family of serine/threonine kinases is responsible for the constitutive phosphorylation of 50% of the total cellular pool of Ii-p35 in a wide variety of APCs, including B lymphocytes, PBMC, immature dendritic cells, and mature dendritic cells. Stimulation of protein kinase C activity in APCs significantly enhanced the kinetics of degradation of class II-associated Ii in Ag-processing compartments and the binding of antigenic peptides to these class II molecules. In cells expressing an Ii-phosphorylation mutant, trafficking of class II molecules to endosomes was impaired and Ii proteolysis was inhibited, demonstrating a direct effect of Ii phosphorylation on MHC class II trafficking. These results demonstrate that phosphorylation of Ii in APCs alters the kinetics of trafficking of newly synthesized class II molecules to lysosomal Ag-processing compartments.