Tetrameric, active PKM2 inhibits IP receptors, potentially requiring GRP75 as an additional interaction partner.

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IPR). This interaction suppresses IPR-mediated cytosolic [Ca] rises. As PKM2 exists in monomeric, dimeric and tetrameric forms displaying different properties including catalytic activity, we investigated the molecular determinants of PKM2 enabling its interaction with IPRs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IPR-mediated Ca signals. Consistently, in PKM2 knock-out HeLa cells, PKM2, a tetrameric, highly active PKM2 mutant, but not inactive PKM2 or the less active PKM2, suppressed IPR-mediated Ca release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IPR1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IPR1), the presumed interaction sites of PKM2 on the IPR. Moreover, on-nucleus patch clamp of heterologously expressed IPR1 in DT40 cells devoid of endogenous IPRs did not reveal any functional effect of purified wild-type PKM2, mutant PKM2 or PKM1 proteins. These results indicate that an additional factor mediates the regulation of the IPR by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IPR1, IPR3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IPR, but also PKM2:GRP75 and GRP75:IPR interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca signaling via the IPR through a multiprotein complex involving GRP75.