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Mapping the site of inositol 1,4,5‐trisphosphate receptor ubiquitination
Author(s) -
Sliter Danielle,
Kirkpatrick Donald,
Alzayady Kamil,
Gygi Stephen
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.605.9
Subject(s) - ubiquitin , endoplasmic reticulum , inositol , receptor , proteasome , microbiology and biotechnology , phospholipase c , chemistry , biochemistry , ubiquitin conjugating enzyme , endogeny , calcium signaling , ubiquitin ligase , biology , gene
Inositol 1,4,5‐trisphosphate (IP3) receptors form tetrameric channels in endoplasmic reticulum (ER) membranes, and mediate IP3‐induced calcium mobilization in mammalian cells. In response to various extracellular stimuli that persistently increase phospholipase C activity, IP3 receptors are ubiquitinated and then degraded by the 26S proteasome. We have previously shown that IP3 receptor ubiquitination is triggered by the combined action of IP3 and calcium, is mediated by the ubiquitin‐conjugating enzyme, mamUbc7, and is processed by the ER‐associated degradation pathway. Here, for endogenous type 1 IP3 receptor processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin chains. Using mass spectrometry we found that a total 43 of the 167 lysines in the receptor are ubiquitinated, with three lysines, K1771, K1884, and K1901, being most frequently identified. Additionally, using ubiquitin‐AQUA analysis we found that most of these sites (~85%) are occupied by monoubiquitin and that both K48 and K63 linkages are abundant in attached chains. Overall, these data show that an endogenous substrate ubiquitinated in situ by endogenous ligases is tagged widely with an array of ubiquitin species. This research was funded by a grant to RJH Wojcikiewicz by the National Institues of Health.