Activated Inositol 145-Trisphosphate Receptors Are Modified by Homogeneous Lys-48- and Lys-63-linked Ubiquitin Chains but Only Lys-48-linked Chains Are Required for Degradation

摘要

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP3 and Ca²⁺-gated Ca²⁺ channels. Endogenous IP3Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP3R1 in αT3-1 pituitary gonadotrophs and found that IP3R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319–35328). Here, we have extended these studies to determine whether IP3R2 and IP3R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP3R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP3Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP3R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP3R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP3Rs may have physiological consequences beyond signaling for degradation.