The specificities of Kaposi's sarcoma-associated herpesvirus-encoded E3 ubiquitin ligases are determined by the positions of lysine or cysteine residues within the intracytoplasmic domains of their targets

Abstract

Kaposi's sarcoma-associated herpesvirus encodes two homologous E3 ligases, MIR1 and MIR2, that mediate the ubiquitination and subsequent downregulation of several cell surface proteins, and in particular major histocompatibility complex class I (MHC-I) molecules. We have previously shown that, in addition to lysine ubiquitination, MIR1 has the unique ability of transferring ubiquitin onto MHC-I molecules lacking available lysine residues, in a cysteine-dependent manner. Here we report that MIR1 activity is maximal when either a lysine or cysteine residue is placed approximately 15 amino acids away from the transmembrane domain, whereas MIR2 preferentially targets residues, including cysteines, that are closer to the transmembrane domain. Thus MIR1 and -2 can distinguish their substrates based on the position of the lysine or cysteine residues, suggesting that these proteins have evolved to target different sets of surface molecules. These results indicate that the position of target residues within a substrate is an essential determinant of E3 ubiquitin ligase specificity.

FIG. 1.

The position of lysine or cysteine residues within the intracytoplasmic tail of HLA.B7 is an essential determinant of MIR activity. (A) BJAB cells stably expressing HLA.B7 with either one lysine or one cysteine at various positions within an artificial glycine/alanine tail were transduced with retroviral vectors expressing MIR1-EGFP or MIR2-EGFP. Downregulation (n-fold) was determined for each HLA.B7 molecule by quantifying surface expression in the presence (GFP⁺) or absence (GFP⁻) of the MIR protein using flow cytometry. (B) BJAB cells stably expressing MIR1 as well as HLA.B7 2R were transfected with either MIR1-EGFP, MIR2-EGFP, or EGFP alone. Surface HLA.B7 expression was analyzed by flow cytometry. Transfected cells are represented by the shaded histogram.

FIG. 2.

Two regions encoded within MIR1 are essential for downregulation of lysine-less HLA.B7 2R. (A) BJAB cells stably expressing wt HLA.B7 or HLA.B7 2R were transfected with chimeric mutants that combine regions of MIR1 and MIR2, and surface expression of HLA.B7 was measured by flow cytometry. (B) Additional chimeras were created in which various portions of the MIR2 N and C termini were placed on a MIR1 background. By determining which chimeras downregulate surface wt HLA.B7 and HLA.B7 2R, one region each within the N and C termini were identified. The alignments of these regions between MIR1 and -2 are shown in panel C.

FIG. 3.

Two regions of the MIR proteins determine the optimal position of the cysteine in the substrate. (A) The two regions of MIR1 identified above were substituted onto MIR2-EGFP to create chimera 2-1-2-1-2. Similarly, the two regions of MIR1 were replaced by the homologous region of MIR2 to create chimera 1-2-1-2-1 fused to EGFP. BJAB cells stably expressing HLA.B7 2R were transfected with vectors expressing either the chimeras or the MIR proteins, and the extent of MHC-I downregulation was assessed by fluorescence-activated cell sorting. (B) BJAB cells stably expressing the HLA.B7 mutants containing cysteines at various positions were transduced with a retroviral vector expressing the chimera 2-1-2-1-2-EGFP and analyzed by flow cytometry as described for Fig. 1.

FIG. 4.

Model illustrating how MIR proteins have evolved to select specific positions to be ubiquitinated within the intracellular regions of their substrates. (A) The MIR proteins interact with their substrates through transmembrane-transmembrane interactions. An additional level of specificity is brought by the position of the lysine or cysteine residue. Whereas MIR1 requires target residues distant from the transmembrane region, MIR2 depends on the presence of lysine or cysteine residues close to the plasma membrane. This constraint is determined by two small regions of the MIR (highlighted in gray) that might orient the positioning of the E2. (B) A schematic drawing of MIR2 showing the relative positions of the N- and C-terminal regions regulating positional constraint (regions A and B) with respect to other motifs previously identified in this molecule (16). RING-CH, RING finger domain; Tyr, tyrosine-based endocytosis motif; SH3B, potential SH3 binding site; DE1 and -2, acidic domains 1 and 2; CM, conserved motif.