title: Palmitoylation of MIR2 is required for its function.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two RING finger E3 ubiquitin ligases (MIR1 and MIR2) that mediate ubiquitination and degradation of cellular proteins important for the establishment of an efficient antiviral immune response. MIR1 and MIR2 share 30% sequence identity; however, their substrate preferences are varied. MIR1 has been shown to primarily downregulate major histocompatibility complex class I (MHC-I), whereas MIR2 can downregulate a wide range of cell surface proteins. Many of the MIR substrates are thought to be present in lipid raft microdomains, a subregion of the plasma membrane known to be important for a wide range of signal transduction events. Palmitoylation is a posttranslational modification that increases recruitment of transmembrane proteins to lipid rafts. In this study, we investigated the importance of palmitoylation for MIR function. We present evidence that MIR2-mediated downregulation of MHC-I and platelet endothelial cell adhesion molecule 1 (PECAM-1) but not other substrates is inhibited in the presence of the drug 2-bromohexadecanoic acid (2-Br), a chemical inhibitor of palmitoylation. Biochemical analysis indicates that MIR2 is directly palmitoylated on cysteine 146. Mutation of this cysteine to a phenylalanine prevents MIR2 palmitoylation and blocks the ability of MIR2 to downregulate MHC-I and PECAM-I but not B7.2 and intercellular adhesion molecule 1 (ICAM-I), consistent with the phenotype observed after 2-Br treatment. Unpalmitoylated MIR2 does not interact with MHC-I and is thus unable to ubiquitinate and downregulate MHC-I from the cell surface. Furthermore, we observed that MIR2 is palmitoylated in vivo during lytic infection. Palmitoylation may act to regulate MIR2 function and localization during viral infection by allowing MIR2 to properly interact with and downregulate multiple substrates known to play an important role in the host immune response.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
authors.forename: Veronica G
authors.initials: VG
authors.lastname: Anania
authors.forename: Laurent
authors.initials: L
authors.lastname: Coscoy
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015536:Down-Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006566:Herpesviridae Infections / Q000378:metabolism / Q000821:virology*; D019288:Herpesvirus 8, Human / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D054878:Lipoylation; D008969:Molecular Sequence Data; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017874:Immediate-Early Proteins
chemical_list: D015395:Histocompatibility Antigens Class I; D044767:Ubiquitin-Protein Ligases; D014764:Viral Proteins; C502006:MIR1 E3 ubiquitin ligase, KSHV; C405867:K5 protein, Human herpesvirus 8; D017874:Immediate-Early Proteins