DEGRADATION OF IMMUNOMODULATORY PROTEINS BY KSHV K5

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanisms involved in targeting cell surface receptors for ubiquitylation are complex. K5 is a MARCH family E3 ubiquitin ligase that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report, we present data that there are multiple molecular mechanisms of ICAM-1, B7.2 and MHC I modulation mediated by this E3 ligase. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct MHC I or ICAM-1 endocytosis, but still able to down regulate B7.2 in a ubiquitindependant, but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. Finally, in addition to its critical role in directing target protein ubiquitylation, confocal microscopy of K5 containing a RING-CH mutant demonstrated that this domain plays a key role in intracellular localization.