Tetrameric, active PKM2 inhibits IP3 receptors, potentially requiring GRP75 as an additional interaction partner

Abstract

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IP₃R). This interaction suppresses IP₃R-mediated cytosolic [Ca²⁺] 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 IP₃Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP₃R-mediated Ca²⁺ signals. Consistently, in PKM2 knock-out HeLa cells, PKM2^C424L, a tetrameric, highly active PKM2 mutant, but not inactive PKM2^K270M or the less active PKM2^K305Q, suppressed IP₃R-mediated Ca²⁺ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP₃R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP₃R1), the presumed interaction sites of PKM2 on the IP₃R. Moreover, on-nucleus patch clamp of heterologously expressed IP₃R1 in DT40 cells devoid of endogenous IP₃Rs 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 IP₃R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP₃R1, IP₃R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP₃R, but also PKM2:GRP75 and GRP75:IP₃R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca²⁺ signaling via the IP₃R through a multiprotein complex involving GRP75.

Keywords: Bcl-2; Ca(2+) signaling; D5SD peptide; GRP75; IP(3) receptor; Pyruvate kinase M2.