Tetrameric, active PKM2 inhibits IP 3 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 (IP ₃ R). This interaction suppresses IP ₃ R-mediated cytosolic [Ca ²⁺ ] 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 IP ₃ Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP ₃ R-mediated Ca ²⁺ 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 IP ₃ R-mediated Ca ²⁺ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP ₃ R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP ₃ R1), the presumed interaction sites of PKM2 on the IP ₃ R. Moreover, on-nucleus patch clamp of heterologously expressed IP ₃ R1 in DT40 cells devoid of endogenous IP ₃ Rs 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 IP ₃ R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP ₃ R1, IP ₃ R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP ₃ R, but also PKM2:GRP75 and GRP75:IP ₃ R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca ²⁺ signaling via the IP ₃ R through a multiprotein complex involving GRP75.
Competing Interests: Declaration of competing interest None.
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