Abstract
Lysophosphatidic acid (LPA) promotes cell survival through the activation of G protein-coupled LPA receptors. However, whether different LPA receptors activate distinct anti-apoptotic signaling pathways is not yet clear. Here we report a novel mechanism by which the LPA(2) receptor targets the proapoptotic Siva-1 protein for LPA-dependent degradation, thereby attenuating Siva-1 function in DNA damage response. The carboxyl-terminal tail of the LPA(2) receptor, but not LPA(1) or LPA(3) receptor, specifically associates with the carboxyl cysteine-rich domain of Siva-1. Prolonged LPA stimulation promotes the association of Siva-1 with the LPA(2) receptor and targets both proteins for ubiquitination and degradation. As a result, adriamycin-induced Siva-1 protein stabilization is attenuated by LPA in an LPA(2)-dependent manner, and the function of Siva-1 in promoting DNA damage-induced apoptosis is inhibited by LPA pretreatment. Consistent with this result, inhibition of the LPA(2) receptor expression increases Siva-1 protein levels and augments adriamycin-induced caspase-3 cleavage and apoptosis. Together, these findings reveal a critical and specific role for the LPA(2) receptor through which LPA directly inactivates a critical component of the death machinery to promote cell survival.
Highlights
Substantial evidence has shown that Lysophosphatidic acid (LPA) can protect cells from serum deprivation-induced apoptosis, prevent chemotherapeutic agents-induced apoptosis, and block death receptor-mediated apoptosis in a variety of cells, suggesting that LPA is a survival factor [7,8,9]
Five mg of total lysates were isolated from DKO-mock and DKO-LPA2 mouse embryonic fibroblasts (MEFs) treated with 20 M MG-132 or not for 2 h, and the LPA2 receptor was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma)
The Carboxyl-terminal Tail of the LPA2 Receptor Interacts with the Carboxyl Cysteine-rich Domain of Siva-1 Protein—In an attempt to identify the molecules involved in the regulation of the LPA2 receptor, a fusion protein containing the carboxyl-terminal tail of the LPA2 receptor and the Gal4 DNA-binding domain was used as bait to screen a HeLa cell cDNA library [14]
Summary
Plasmid Construction—To construct the Siva-1 expression vector, a human Siva-1 cDNA isolated from a yeast two-hybrid screen, which contains six base pairs 5Ј to the translation start sites, the entire Siva-1 coding sequences and part of the 3Ј-untranslated region, was cloned into pcDNA3 for the expression of full-length Siva-1. Semiquantitative RT-PCR analysis was performed to select cell lines expressing FLAG-LPA2 mRNA at the levels comparable with the endogenous LPA2 in wild-type MEFs. Five mg of total lysates were isolated from DKO-mock and DKO-LPA2 MEFs treated with 20 M MG-132 or not for 2 h, and the LPA2 receptor was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma). FLAG-tagged Siva-1 or LPA2 receptor was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma), and the immunoblot was probed with an anti-HA polyclonal antibody (Santa Cruz Biotechnology) to detect ubiquitinylated proteins. FLAG-Siva-1 in the whole cell lysates and the immunoprecipitated FLAG-LPA2 receptor were detected by immunoblotting using an anti-FLAG polyclonal antibody (Sigma). Apoptosis was determined by annexin V-fluorescein isothiocyanate staining (BD Biosciences) followed by fluorescence-activated cell sorter analysis
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