The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp.
The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63-linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63-linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.
Tuvia
Shmuel
S
Proteologics Ltd., Kiryat Weizmann, Rehovot 76124, Israel.
Taglicht
Daniel
D
Erez
Omri
O
Alroy
Iris
I
Alchanati
Iris
I
Bicoviski
Vivian
V
Dori-Bachash
Mally
M
Ben-Avraham
Danny
D
Reiss
Yuval
Y
D002118:Calcium / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006706:Homeostasis; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D019284:Thapsigargin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*; D021601:trans-Golgi Network / Q000378:metabolism
D004791:Enzyme Inhibitors; C485932:HERPUD1 protein, human; D008565:Membrane Proteins; D025801:Ubiquitin; D014415:Tunicamycin; D019284:Thapsigargin; C498193:SH3RF1 protein, human; D044767:Ubiquitin-Protein Ligases; D002118:Calcium